Infectious clone of human parvovirus b19 and methods

ABSTRACT

The invention relates to infectious clones of parvovirus B19, methods of cloning infectious B19 clones, and methods of cloning viral genomes that have secondary DNA structures that are unstable in bacterial cells. A B19 infectious clone and methods of producing B19 infectious clones are useful for producing infectious virus. Infectious virus is useful for identifying and developing therapeutically effective compositions for treatment and/or prevention of human parvovirus B19 infections.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Part of the work performed during the development of this inventionutilized United States government funds under the Division of IntramuralResearch, NHLBI.NIH.

BACKGROUND OF THE INVENTION

Human parvovirus B19 is the only member of the Parvoviridae family knownto cause diseases in humans. Parvovirus B19 infection causes fifthdisease in children, polyarthropathy syndromes in adults, transientaplastic crisis in patients with underlying chronic hemolytic anemia,and chronic anemia due to persistent infection in immunocompromisedpatients. Hydrops fetalis and fetal death have been reported aftermaternal infection with parvovirus B19 during pregnancy (Brown et al.,1994, Crit. Rev. Oncol./Hematol. 16:1-13).

Parvovirus B19 exhibits a selective tropism for erythroid progenitorcells. The virus can be cultured in erythroid progenitor cells from bonemarrow, fetal liver cells, and cell lines such as UT7/Epo or KU812Ep6.(Ozawa et al., 1986, Science 233:883-886; Brown et al., 1991, J. Gen.Vir. 72:741-745; Komatsu et al., 1993, Blood 82:456-464; Shimomura etal., 1992, Blood 79:18-24; Miyagawa et al., 1999, J. Virol. Methods83:45-54). Although the virus can be cultured in these cells very littlevirus is produced. The selective tropism of the virus is mediated inpart by neutral glycolipid globoside (blood group P antigen), which ispresent on cells of the erythroid lineage (Brown et al., 1993, Science,262:114-117). The presence of globoside on the surface of a cell is adeterminant of viral tropism. Parvovirus B19 has a cytotoxic effect onerythroid progenitor cells in bone barrow and causes interruption oferythrocyte production. Human bone marrow cells that lack globoside onthe cell surface are resistant to parvovirus B19 infection (Brown etal., 1994, N. Engl. J. Med., 33:1192-1196).

The ends of the parvovirus B19 genome have long inverted repeats (ITR),which are imperfect palindromes that form double-stranded hairpins. Therole of the ITRs in the parvovirus B19 viral life cycle is unknown dueto the inability to produce an infectious clone containing complete ITRsequences. In other parvoviruses, ITRs play an important role in theviral life cycle: they serve as primers for the synthesis of thecomplementary strand of viral DNA and are essential for the replication,transcription, and packaging of virus DNA (Berns, K (1990) in Virology,eds. Fields et al. Raven Press Ltd, NY, pp 1743-1763). Previous attemptsto produce an infectious clone of parvovirus B19 were unsuccessful dueto deletions in the ITR sequences and the instability of the ITRs inbacterial cells (Deiss et al., 1990, Virology 175:247-254; Shade et al.,1986, J. Virol. 58:921-936). Methods of consistently producinginfectious B19 parvovirus in cell culture are not known.

Thus, there remains a need to develop an infectious clone of parvovirusB19. A B19 infectious clone and methods of producing B19 infectiousclones can be useful for producing infectious virus. Infectious virus isuseful for identifying and developing therapeutically effectivecompositions for treatment and/or prevention of human parvovirus B19infections, such as for example, antibodies, attenuated vaccines, andchimeric viral capsid proteins comprising antigenic epitopes.

SUMMARY OF THE INVENTION

One aspect of the invention is directed to methods of cloning aparvovirus B19 viral genome. Clones of viral genomes produced by themethods of the invention are useful for consistently producinginfectious virus. Infectious virus is useful for identifying anddeveloping therapeutically effective compositions for treatment and/orprevention of human parvovirus B19 infections, such as for example,antibodies, attenuated vaccines, and chimeric viral capsid proteinscomprising antigenic epitopes.

The methods of cloning a parvovirus B19 viral genome generally employintroducing a vector comprising all or a portion of a parvovirus B19genome into a prokaryotic cell that is deficient in at least onerecombinase enzyme; incubating the cells at about 25° C. to 35° C.; andrecovering the vector from the prokaryotic cells. An inverted terminalrepeat (ITR) may be at the 5′ end or 3′ end or both of the viral genome.In an embodiment, the ITR comprises a nucleic acid sequence of SEQ IDNO:1 or SEQ ID NO:2. In addition to at least one ITR, the viral genomemay comprise a nucleic acid sequence encoding at least one or all ofVP2, nonstructural protein (NS), or 11-kDa protein. The bacterial cellmay be recA1, endA, recB, and/or recJ deficient. In an embodiment, thebacterial cell comprises a genotype of e14-(McrA-)Δ(mcrCB-hsdSMR-mrr)171endA1 supE44 thi-1 gyrA96 relA1 lac recB recJ sbcC umuC::Tn5 (Kanr) uvrC[F′ proAB lacIqZ.M15 Tn10 (Tetr)]. Vectors that are useful in themethods of the invention include pBR322, p ProExHTb, pUc19 andpBluescript SK.

In some embodiments, the full length B19 genome is cloned by cloning atleast two portions of the viral genome into separate vectors andrecombining the two portions into a single vector. Preferably, twoportions of the viral genome comprise an ITR at the end of the portion.The portions of the viral genome can be obtained by digesting the genomewith a restriction enzyme that cuts the genome at a location between theITRs. Preferably the restriction enzyme cuts the genome at a location atleast about 800 nucleotides from the ITR. The portions may be cut andreligated to reduce the vector size and eliminate undesired restrictionsites. For example, the B19 genome may be digested with BamHI. The twofragments (right end genome fragment and left end genome fragment)generated by BamHI digestion are ligated into separate vectors and thefull-length genome is generated by recombining the right end genomefragment and left end genome fragment into a single vector. In anembodiment, the full length genome comprises a nucleic acid sequence ofSEQ ID NO:5.

The methods of producing or identifying an infectious clone orinfectious virus of parvovirus B19 generally employ introducing a vectorcomprising all or a portion of a viral genome of parvovirus B19 into apopulation of cells, wherein the vector is present in at least about 15%of the cells; and incubating the cells under conditions to allow forviral replication. Preferably, the cells are eukaryotic cells, morepreferably permissive cells such as for example erythroid progenitorcells, fetal liver cells, UT7/EPO cells, UT7/EPO-S1 cells, or KU812Ep6cells. In some embodiments, the cells are cultured in vitro. Optionally,viral replication can be detected in the cells.

The vector may be introduced into the cells using standard transfectiontechniques known in the art. In an embodiment, the cells are transfectedby electroporation or electrical nuclear transport. The viral genomepreferably comprises an ITR sequence having a nucleic acid sequence ofSEQ ID NO:1 or SEQ ID NO:2 and a nucleic acid sequence encoding one ormore of 11-kDa protein, NS protein, VP1, VP2, or putative protein X.Preferably the ITRs are located at the 5′ end or 3′ end of the genome.In an embodiment, the infectious clone comprises a polynucleotidenucleic acid sequence of SEQ ID NO:5. Reproduction of the infectiousclones produced by the methods of the invention can be detected bycontacting permissive cells with supernatant from the population ofcells and analyzing the contacted cells for spliced capsid transcriptsor capsid proteins.

Another aspect of the invention is directed to isolated infectiousparvovirus B19 clones. The clones may be produced by the methods of theinvention. Infectious B19 clones are useful in diagnostic assays,identifying and developing therapeutically effective compositions fortreatment and/or prevention of human parvovirus B19 infections, such asfor example, antibodies, attenuated vaccines, and chimeric viral capsidproteins comprising antigenic epitopes. Preferably the clones compriseall or a portion of a parvovirus B19 genome and a replicable vector. Thegenome may comprise ITRs located at the 5′ end and 3′ end of the genomeand a nucleic acid sequence encoding one or more of VP1, VP2, NS, 11-kDaprotein, 7.5-kDa protein, or putative protein X. Preferably the viralgenome comprises a polynucleotide having at least 90% nucleic acidsequence identity to SEQ ID NO:5 or SEQ ID NO:24. In an embodiment, theviral genome comprises a nucleotide sequence of SEQ ID NO:5.

The invention also encompasses using the infectious parvovirus B19 cloneof the invention and/or host cells comprising the clone as immunogeniccompositions to prepare vaccine components and/or to develop antibodiesthat can be used in diagnostic assays or to inhibit or antagonize B19infection of cells. Host cell cultures comprising the parvovirus B19clone can be heat inactivated and used as an immunogen. Passaging of aninfectious clone in vitro can provide an attenuated strain of parvovirusB19 useful in vaccine compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a map of recombinant plasmid pB19-N8, which contains aninsert comprising 4844 nucleotides of the B19 genome. The arrowsindicate genes. Shaded arrows indicate genes in the B19 genome. Theshaded circles at the 5′ and 3′ ends of the B19 genome indicate the ITRsequences.

FIG. 2A shows the structure of the B19 terminal inverted repeats inhairpin form. The “flip” (SEQ ID NO:1) and “flop” (SEQ ID NO:2)orientations at the 5′ end (+ strand) are shown.

FIG. 2B shows a comparison of nucleic acid sequences encoding the flipand flop forms of ITRs in B19 isolate J35 (SEQ ID NOS: 1 and 2), the B19isolate reported by Deiss et al., 1990, Virology, 175:247-254 (SEQ IDNOS:3 and 4), and the flop form in B19-Hv (SEQ ID NO:37). Alignedpositions are boxed in black. The numbering indicates the positions ofnucleotides in the genomes of the respective B19 isolates.

FIG. 3 shows the experimental strategy used to construct an infectiousclone of parvovirus B19.

FIG. 4 shows a map of recombinant plasmid pB19-4244, which contains aninsert comprising 5592 nucleotides of full-length B19 genome (SEQ IDNO:5). The arrows indicate genes. Shaded arrows indicate genes in theB19 genome. The shaded circles at the 5′ and 3′ ends of the B19 genomeindicate the ITR sequences.

FIG. 5 shows a map of recombinant plasmid pB19-4244d. The arrowsindicate genes. The shaded circles at the 5′ and 3′ ends of the B19genome indicate the ITR sequences. Shaded arrows indicate genes in theB19 genome. Plasmid pB19-4244d was modified from pB19-4244 byXhoI-Ecl36II digestion to remove the undesired XbaI site.

FIG. 6 shows a map of recombinant plasmid pB19-M20. The arrows indicategenes. Shaded arrows indicate genes in the B19 genome. The shadedcircles at the 5′ and 3′ ends of the B19 genome indicate the ITRsequences. Nucleic acid residue 2285 was substituted (C2285T) generatinga DdeI site in the B19 genome.

FIG. 7 shows a schematic representation of the replication of B19 viralgenome. The replicative DNA form provides evidence of viral DNAreplication and can be distinguished by BamHI restriction enzymedigestion.

FIG. 8 shows RT-PCR analysis of UT7/Epo-S1 cells for B19 transcripts.The cells were transfected with recombinant plasmids or infected withB19 virus. Total RNA was extracted from the cells 72 h post-transfectionor 72 h post-infection. RT-PCR was performed with a primer pair of B19-1(SEQ ID NO:6) and B19-9 (SEQ ID NO:7). The PCR products were separatedby agarose electrophoresis and analyzed by Southern blotting with analkaline-phosphatase-labeled probe. (+) and (−) indicate the presence orabsence respectively of reverse transcriptase in the PCR reaction. Thenumbers with arrows indicate amplicon size in base pairs (bp).

FIGS. 9A-C show detection of B19 capsid proteins in UT7/Epo-S1 cellsinfected with B19 virus (FIG. 9A), UT7/Epo-S1 cells transfected withpB19-M20 (FIG. 9B), and UT7/Epo-S1 cells transfected with pB19-N8 (FIG.9C). The B19 capsid proteins were detected 72 h post-transfection or 72h post-infection using monoclonal antibody 521-5D (gift from Dr. LarryAnderson, Centers for Disease Control and Prevention, Atlanta, Ga.).Magnification is 750×.

FIG. 10 shows Southern blot analysis of DNA purified from cellstransfected with SalI digested fragment of pB19-M20 or pB19-4244. DNAfrom B19 virus was used as a positive control. The purified DNA wasdigested with BamHI or EcoRI and the fragments separated by agaroseelectrophoresis. The fragments were probed with a ³²P-random-primedprobed of the complete B19 coding region. Distinct doublets of 1.5 and1.4 kb were detected in transfected cell samples digested with BamHI,but not in the plasmid controls. The 1.4 kb band is a definitive markerfor viral genome replication.

FIG. 11 shows Southern blot analysis of DNA purified from cellstransfected with undigested pB19-M20 or pB19-4244. The purified DNA wasdigested with BamHI or EcoRI and the fragments separated by agaroseelectrophoresis. The fragments were detected with a ³²P-random-primedprobe of the complete B19 coding region. Distinct doublets of 1.5 and1.4 kb were detected in transfected cell samples digested with BamHI.The 1.4 kb band is a definitive marker for viral genome replication. Aband with a molecular size of 5.6 kb, which corresponds to the size ofthe B19 genome, was also detected in EcoRI digested DNA. The 5.6 kb bandindicated that progeny viral DNA was produced by the transfected cellsas neither the B19 genome nor the vector contained an EcoRI restrictionenzyme site.

FIGS. 12A and 12B show RT-PCR analysis of UT7/Epo-S1 cells infected withclarified supernatant from B19-infected or p19-4244, pB19-M20, orpB19-N8 transfected cells for B19 transcripts. Total RNA was extractedfrom the cells 0 h (FIG. 12A) and 72 h (FIG. 12B) post-infection. RT-PCRwas performed with a primer pair of B19-1 (SEQ ID NO:6) and B19-9 (SEQID NO:7). The PCR products were separated by agarose electrophoresis andanalyzed by Southern blotting with an alkaline-phosphatase-labeledprobe. (+) and (−) indicate the presence or absence respectively ofreverse transcriptase in the PCR reaction. The numbers with arrowsindicate amplicon size in base pairs (bp).

FIGS. 13A-C show detection of B19 capsid proteins in cells infected withclarified supernatant from B19-infected (FIG. 13A), or pB19-M20 (FIG.13B) or pB19-N8 (FIG. 13C) transfected cells. B19 capsid proteins weredetected 72 h post-infection using monoclonal antibody 521-5D.Magnification is 750×.

FIG. 14 shows a comparison of a portion of nucleic acid sequence fromB19 clone J35 and B19 clone M20. M20 virus has a DdeI restriction sitethat is not present in J35 virus.

FIG. 15 shows RT-PCR analysis of B19 transcripts in UT7/Epo-S1 cellsinfected with J35 virus or infectious clone pB19-M20. cDNA derived fromthe infected cells was amplified using a primer pair of B19-2255 (SEQ IDNO:8) and B19-2543 (SEQ ID NO:9). The PCR products were digested withDdeI and analyzed by gel electrophoresis. (+) and (−) indicate thepresence or absence respectively of reverse transcriptase in the PCRreaction. The numbers with arrows indicate amplicon size in base pairs(bp).

FIG. 16A-F shows RT-PCR analysis of B19 transcripts in UT7/Epo-S1 cellstransfected with pB19-M20 (FIG. 16A), pB19-M20/NS (FIG. 16A),pB19-M20/VP1(−) (FIG. 16B), pB19-M20/11(−) (FIG. 16C), pB19-M20/7.5(−)(FIG. 16D), pB19-M20/X(−) (FIG. 16E), or pB19-N8 (FIG. 16F). At 72 hpost-transfection, cells were infected with clarified supernatant fromthe transfected cells. Total RNA was extracted from the cells 72 hpost-tranfection or 72 h post-infection. RT-PCR was performed with aprimer pair of B19-1 (SEQ ID NO:6) and B19-9 (SEQ ID NO:7). The PCRproducts were separated by gel electrophoresis. (+) and (−) indicate thepresence or absence respectively of reverse transcriptase in the PCRreaction.

FIGS. 17A-D show detection of B19 capsid proteins and 11-kDa protein incells transfected with pB19-M20 (FIGS. 17A and 17B respectively) orpB19-M20/11(−) (FIGS. 17C and 17D respectively). B19 capsid proteinswere detected 72 h post-infection using monoclonal antibody 521-5D (FIG.17A; 17C). 11-kDa protein was detected 72 h post-transfection using arabbit polyclonal anti-11-kDa protein antibody (FIGS. 17B, 17D).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “antibody” is used in the broadest sense and specificallyincludes, for example, single anti-parvovirus B19 monoclonal antibodies,anti-parvovirus B19 antibody compositions with polyepitopic specificity,single chain anti-parvovirus B19 antibodies, and fragments ofanti-parvovirus B19 antibodies. The term “monoclonal antibody” as usedherein refers to an antibody obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., 1995, ProteinEng., 8:1057-1062); single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

The term “binds specifically” refers to an antibody that bindsparvovirus B19 and does not substantially bind other parvoviruses. Insome embodiments, the antibody specifically binds a first B19 isolateand does not bind a second B19 isolate. For example, an antibody mayspecifically bind B19-Au and not bind B19-HV.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers, which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations, employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

The term “parvovirus B19”, “B19”, “B19 virus”, “B19 clone”, or “B19isolate” means an isolate, clone or variant B19 viral genome ofparvovirus B19 of the family Parvoviridae including genotypes 1, 2, and3. A naturally occurring isolate of parvovirus B19 of the invention hasat least 90% nucleic acid identity to human parvovirus B19-Au (GenBankaccession number M13178; SEQ ID NO:24), which lacks intact ITRs at both5′ and 3′ ends of the genome (Shade et al., 1986, J. Virol.,58:921-936). B19 has a non-enveloped, icosahedral capsid packaging asingle-stranded DNA genome of approximately 5600 nucleotides.Transcription of the B19 genome is controlled by the single promoter p6located at map unit 6, which regulates the synthesis of viral proteinsincluding, but not limited to, nonstructural protein (NS), capsidproteins VP1 and VP2, 11-kDa protein, 7.5-kDa protein, and putativeprotein X. B19 viral DNA can be isolated from infected humans or cellsor can be prepared as described herein. An embodiment of an isolate ofparvovirus B19 has a nucleotide sequence of SEQ ID NO:5 (Table 1). Insome embodiments, the B19 genome cloned into the vector may have from 1to about 5 nucleotides deleted from the 5′ end and/or 3′ end of the fulllength viral genome. For example, the B19 genome (SEQ ID NO:5) clonedinto pB19-4244 (FIG. 4) has 2 nucleic acids deleted from the 5′ end and3′ end compared to the nucleic acid sequence of the full length genome(SEQ ID NO:38).

TABLE 1    1   aaatcaga tgccgccggt cgccgccggt aggcgggact tccggtacaagatggcggac   59 aattacgtca tttcctgtga cgtcatttcc tgtgacgtca cttccggtgggcgggacttc  119 cggaattagg gttggctctg ggccagcttg cttggggttg ccttgacactaagacaagcg  179 gcgcgccgct tgatcttagt ggcacgtcaa ccccaagcgc tggcccagagccaaccctaa  239 ttccggaagt cccgcccacc ggaagtgacg tcacaggaaa tgacgtcacaggaaatgacg  299 taattgtccg ccatcttgta ccggaagtcc cgcctaccgg cggcgaccggcggcatctga  359 tttggtgtct tcttttaaat tttagcgggc ttttttcccg ccttatgcaaatgggcagcc  419 attttaagtg ttttactata attttattgg tcagttttgt aacggttaaaatgggcggag  479 cgtaggcggg gactacagta tatatageac agcactgccg cagctctttctttctgggct  539 gctttttcct ggactttctt gctgtttttt gtgagctaac taacaggtatttatactact  599 tgttaatata ctaacatgga gctatttaga ggggtgcttc aagtttcttctaatgttctg  659 gactgtgcta acgataactg gtggtgctct ttactagatt tagacacttctgactgggaa  719 ccactaactc atactaacag actaatggca atatacttaa gcagtgtggcttctaagctt  779 gaccttaccg gggggccact agcagggtgc ttgtactttt ttcaagcagaatgtaacaaa  839 tttgaagaag gctatcatat tcatgtggtt attggggggc cagggttaaaccccagaaac  899 ctcacagtgt gtgtagaggg gttatttaat aatgtacttt atcactttgtaactgaaaat  959 gtgaagctaa aatttttgcc aggaatgact acaaaaggca aatactttagagatggagag 1019 cagtttatag aaaactattt aatgaaaaaa atacctttaa atgttgtatggtgtgttact 1079 aatattgatg gatatataga tacctgtatt tctgctactt ttagaaggggagcttgccat 1139 gccaagaaac cccgcattac cacagccata aatgatacta gtagcgatgctggggagtct 1199 agcggcacag gggcagaggt tgtgccattt aatgggaagg gaactaaggctagcataaag 1259 tttcaaacta tggtaaactg gttgtgtgaa aacagagtgt ttacagaggataagtggaaa 1319 ctagttgact ttaaccagta cactttacta agcagtagtc acagtggaagttttcaaatt 1379 caaagtgcac taaaactagc aatttataaa gcaactaatt tagtgcctactagcacattt 1439 ttattgcata cagactttga gcaggttatg tgtattaaag acaataaaattgttaaattg 1499 ttactttgtc aaaactatga ccccctattg gtggggcagc atgtgttaaagtggattgat 1559 aaaaaatgtg gcaagaaaaa tacactgtgg ttttatgggc cgccaagtacaggaaaaaca 1619 aacttggcaa tggccattgc taaaagtgtt ccagtatatg gcatggttaactggaataat 1679 gaaaactttc catttaatga tgtagcagga aaaagcttgg tggtctgggatgaaggtatt 1739 attaagtcta caattgtaga agctgcaaaa gccattttag gcgggcaacccaccagggta 1799 gatcaaaaaa tgcgtggaag tgtagctgtg cctggagtac ctgtggttataaccagcaat 1859 ggtgacatta cttttgttgt aagcgggaac actacaacaa ctgtacatgctaaagcctta 1919 aaagagcgca tggtaaagtt aaactttact gtaagatgca gccctgacatggggttacta 1979 acagaggctg atgtacaaca gtggcttaca tggtgtaatg cacaaagctgggaccactat 2039 gaaaactggg caataaacta cacttttgat ttccctggaa ttaatgcagatgccctccac 2099 ccagacctcc aaaccacccc aattgtcaca gacaccagta tcagcagcagtggtggtgaa 2159 agctctgaag aactcagtga aagcagcttt tttaacctca tcaccccaggcgcctggaac 2219 actgaaaccc cgcgctctag tacgcccatc cccgggacca gttcaggagaatcatttgtc 2279 ggaagcccag tttcctccga agttgtagct gcatcgtggg aagaagccttctacacacct 2339 ttggcagacc agtttcgtga actgttagtt ggggttgatt atgtgtgggacggtgtaagg 2399 ggtttacctg tgtgttgtgt gcaacatatt aacaatagtg ggggaggcttgggactttgt 2459 ccccattgca ttaatgtagg ggcttggtat aatggatgga aatttcgagaatttacccca 2519 gatttggtgc gatgtagctg ccatgtggga gcttctaatc ccttttctgtgctaacctgc 2579 aaaaaatgtg cttacctgtc tggattgcaa agctttgtag attatgagtaaagaaagtgg 2639 caaatggtgg gaaagtgatg atgaatttgc taaagctgtg tatcagcaatttgtggaatt 2699 ttatgaaaag gttactggaa cagacttaga gcttattcaa atattaaaagatcattataa 2759 tatttcttta gataatcccc tagaaaaccc atcctctctg tttgacttagttgctcgcat 2819 taaaaataac cttaaaaatt ctccagactt atatagtcat cattttcaaagtcatggaca 2879 gttatctgac cacccccatg ccttatcatc cagtagcagt catgcagaacctagaggaga 2939 agatgcagta ttatctagtg aagacttaca caagcctggg caagttagcgtacaactacc 2999 cggtactaac tatgttgggc ctggcaatga gctacaagct gggcccccgcaaagtgctgt 3059 tgacagtgct gcaaggattc atgactttag gtatagccaa ctggctaagttgggaataaa 3119 tccatatact cattggactg tagcagatga agagctttta aaaaatataaaaaatgaaac 3179 tgggtttcaa gcacaagtag taaaagacta ctttacttta aaaggtgcagctgcccctgt 3239 ggcccatttt caaggaagtt tgccggaagt tcccgcttac aacgcctcagaaaaataccc 3299 aagcatgact tcagttaatt ctgcagaagc cagcactggt gcaggaggggggggcagtaa 3359 tcctgtcaaa agcatgtgga gtgagggggc cacttttagt gccaactctgtgacttgtac 3419 attttctaga cagtttttaa ttccatatga cccagagcac cattataaggtgttttctcc 3479 cgcagcaagt agctgccaca atgccagtgg aaaggaggca aaggtttgcaccattagtcc 3539 cataatggga tactcaaccc catggagata tttagatttt aatgctttaaacttattttt 3599 ttcaccttta gagtttcagc acttaattga aaattatgga agtatagctcctgatgcttt 3659 aactgtaacc atatcagaaa ttgctgttaa ggatgttaca gacaaaactggagggggggt 3719 gcaggttact gacagcacta cagggcgcct atgcatgtta gtagaccatgaatacaagta 3779 cccatatgtg ttagggcaag gtcaagatac tttagcccca gaacttcctatttgggtata 3839 ctttccccct caatatgctt acttaacagt aggagatgtt aacacacaaggaatttctgg 3899 agacagcaaa aaattagcaa gtgaagaatc agcattttat gttttggaacacagttcttt 3959 tcagctttta ggtacaggag gtacagcaac tatgtcttat aagtttcctccagtgccccc 4019 agaaaattta gagggctgca gtcaacactt ttatgagatg tacaatcccttatacggatc 4079 ccgcttaggg gttcctgaca cattaggagg tgacccaaaa tttagatctttaacacatga 4139 agaccatgca attcagcccc aaaacttcat gccagggcca ctagtaaactcagtgtctac 4199 aaaggaggga gacagctcta atactggagc tgggaaagcc ttaacaggccttagcacagg 4259 tacctctcaa aacactagaa tatccttacg cccggggcca gtgtctcagccgtaccacca 4319 ctgggacaca gataaatatg tcacaggaat aaatgctatt tctcatggtcagaccactta 4379 tggtaacgct gaagacaaag agtatcagca aggagtgggt agatttccaaatgaaaaaga 4439 acagctaaaa cagttacagg gtttaaacat gcacacctac tttcccaataaaggaaccca 4499 gcaatataca gatcaaattg agcgccccct aatggtgggt tctgtatggaacagaagagc 4559 ccttcactat gaaagccagc tgtggagtaa aattccaaat ttagatgacagttttaaaac 4619 tcagtttgca gccttaggag gatggggttt gcatcagcca cctcctcaaatatttttaaa 4679 aatattacca caaagtgggc caattggagg tattaaatca atgggaattactaccttagt 4739 tcagtatgcc gtgggaatta tgacagtaac catgacattt aaattggggccccgtaaagc 4799 tacgggacgg tggaatcctc aacctggagt atatcccccg cacgcagcaggtcatttacc 4859 atatgtacta tatgacccta cagctacaga tgcaaaacaa caccacagacatggatatga 4919 aaagcctgaa gaattgtgga cagccaaaag ccgtgtgcac ccattgtaaacactccccac 4979 cgtgccctca gccaggatgc gtaactaaac gcccaccagt accacccagactgtacctgc 5039 cccctcctat acctataaga cagcctaaca caaaagatat agacaatgtagaatttaagt 5099 atttaaccag atatgaacaa catgttatta gaatgttaag attgtgtaatatgtatcaaa 5159 atttagaaaa ataaacgttt gttgtggtta aaaaattatg ttgttgcgctttaaaaattt 5219 aaaagaagac accaaatcag atgccgccgg tcgccgccgg taggcgggacttccggtaca 5279 agatggcgga caattacgtc atttcctgtg acgtcatttc ctgtgacgtcacttccggtg 5339 ggcggaactt ccggaattag ggttggctct gggccagcgc ttggggttgacgtgccacta 5399 agatcaagcg gcgcgccgct tgtcttagtg tcaaggcaac cccaagcaagctggcccaga 5459 gccaacccta attccggaag tcccgcccac cggaagtgac gtcacaggaaatgacgtcac 5519 aggaaatgac gtaattgtcc gccatcttgt accggaagtc ccgcctaccggcggcgaccg 5579 gcggcatctg attt

“Variants” of the parvovirus B19 viral genome refer to a sequence of aviral genome that differs from a reference sequence and includes“naturally occurring” variants as well as variants that are prepared byalteration of one more nucleotides. In some embodiments, when the viralgenome has the sequence of a naturally occurring isolate, the referencesequence may be human parvovirus B19-Au (GeneBank accession numberM13178; SEQ ID NO:24), which lacks intact ITRs at both 5′ and 3′ ends ofthe genome and the variant has at least 90% sequence identity to thereference sequence. In other cases, a variant may be prepared byaltering or modifying the nucleic acid sequence of the viral genomeincluding by addition, substitution, and deletion of nucleotides. Inthat case, the reference sequence can be that of parvovirus B19comprising a polynucleotide sequence of SEQ ID NO:5. In someembodiments, a parvovirus genome has at least 90% sequence identity,more preferably at least 91%, more preferably at least 92%, morepreferably at least 93%, more preferably at least 94%, more preferablyat least 95%, more preferably at least 96%, more preferably at least97%, more preferably at least 98%, more preferably at least 99% orgreater sequence identity to that of a parvovirus B19 genome comprisinga nucleic acid sequence of parvovirus B19 Au (GeneBank accession numberM13178; SEQ ID NO:24) or a parvovirus B19 comprising a polynucleotidesequence of SEQ ID NO:5.

An “infectious clone” of parvovirus B19 as used herein refers to afull-length genome or portion of a genome of a parvovirus B19 isolatecloned into a replicable vector that provides for amplification of theviral genome in a cell. In some embodiments, a portion of the parvovirusB19 genome comprises or consists of nucleic acid sequence encoding atleast one ITR, VP2, NS, and 11-kDa in a single replicable vector. Inother embodiment, the viral genome is a full-length genome. Thereplicable vector provides for introduction and amplification of theviral genome in a wide variety of prokaryotic and eukaryotic cells

The term “erythroid progenitor cell” as used herein refers to a redblood cell precursor cell that differentiates to produce red bloodcells.

“Electrical nuclear transport” is a method of introducing nucleic acidsinto cells such as eukaryotic cells using an electrical current. In someembodiments, in electrical nuclear transport, a recombinant plasmid istransported into the nucleus of cells. Greater amounts of DNA aretransported into the nucleus of dividing cells with electrical nucleartransport than may be expected by cell division alone, therebysubstantially increasing the likelihood of integration of completeexpression cassettes. Electrical nuclear transport methods and buffersystems are described in U.S. 20040014220.

The term “full length genome” refers to a complete coding sequence of aviral genome that comprises at least 75% or greater of the nucleotidesequence that forms the hairpin of the ITR at the 5′ end and 3′ end ofthe genome. In an embodiment, the coding sequence comprises nucleic acidsequence encoding VP1, VP2, NS, 11-kDa protein, 7.5-kDa protein, andputative protein X. In another embodiment, ITRs at each end of the fulllength clone may have 1 to about 5 deletions at each end and retain theability to provide for replication and expression of viral proteins. Inpreferred embodiments, the ITR has at least about 94%, more preferably95%, more preferable 96%, more preferably 97%, more preferably 98%, morepreferably about 99%, and more preferably 100% of the sequence of thatof viral genome isolated from nature, such as that of SEQ ID NO:5 or SEQID:24.

The terms fusion protein” and a “fusion polypeptide” refer to apolypeptide having two portions covalently linked together, where eachof the portions is a polypeptide having a different property. Theproperty may be a biological property, such as activity in vitro or invivo. The property may also be a simple chemical or physical property,such as binding to a target molecule, catalysis of a reaction, etc. Thetwo portions may be linked directly by a single peptide bond or througha peptide linker containing one or more amino acid residues. Generally,the two portions and the linker will be in reading frame with eachother.

The term “infection” as used herein refers to the introduction B19 viralDNA into a cell wherein introduction of the viral DNA into the cell ismediated by B19 capsid. Cells are typically infected by contacting thecell with B19 virus. Infection of a cell by B19 virus may be determinedby analyzing the cell for increase in viral DNA including by detectingthe presence or increase of spliced capsid transcripts and/or unsplicedNS transcripts and/or capsid proteins.

The term “immunogenic effective amount” of a parvovirus B19 or componentof a parvovirus refers to an amount of a parvovirus B19 or componentthereof that induces an immune response in an animal. The immuneresponse may be determined by measuring a T or B cell response.Typically, the induction of an immune response is determined by thedetection of antibodies specific for parvovirus B19 or componentthereof.

An “isolated” antibody is an antibody that has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source.Preferably, the isolated nucleic is free of association with allcomponents with which it is naturally associated. An isolated nucleicacid molecule is other than in the form or setting in which it is foundin nature. Isolated nucleic acid molecules encoding, for example, B19genome or B19 viral proteins therefore are distinguished from nucleicacid molecules encoding B19 viral proteins or a B19 genome as it mayexist in nature. In an embodiment, the B19 genome comprises a nucleicacid sequence encoding one or more of 11-kDa protein, VP1, VP2, NS,7.5-kDa protein, and protein X. In another embodiment, thepolynucleotides have a nucleotide sequence that encodes a B19 genomethat has greater than 99% nucleic acid sequence identity to SEQ ID NO:5(Table 1). Preferably, the polynucleotide encodes an infectious clone ofparvovirus B19.

“ITR” or “ITR sequence” refers to an inverted terminal repeat ofnucleotides in a nucleic acid such as a viral genome. The ITRs includean imperfect palindrome that allows for the formation of a doublestranded hairpin with some areas of mismatch that form bubbles. The ITRsserve as a primer for viral replication and contain a recognition sitefor NS protein that may be required for viral replication andassembling. In some embodiments, the location and number of the bubblesor areas of mismatch are conserved as well as the NS binding site. TheNS binding site provides for cleavage and replication of the viralgenome. In an embodiment, the parvovirus B19 genome comprises one ormore ITR sequences. Preferably, the B19 genome comprises an ITR sequenceat the 5′ end and the 3′ end. An ITR may be about 350 nucleotides toabout 400 nucleotides in length. An imperfect palindrome may be formedby about 350 to about 370 of the distal nucleotides, more preferablyabout 360 to about 365 of the distal nucleotides. Preferably theimperfect palindrome forms a double-stranded hairpin. In an embodiment,the ITRs are about 383 nucleotides in length, of which about 365 of thedistal nucleotides are imperfect palindromes that form double-strandedhairpins. In another embodiment, the ITRs are about 381 nucleotides inlength, of which about 361 of the distal nucleotides are imperfectpalindromes that form double-stranded hairpins. In some embodiments, aB19 genome comprises at least 75% of the nucleotide sequence that formsthe hairpin in the ITR at the 5′ end and 3′ end of the genome. In otherembodiments, the ITRs may have 1 to about 5 nucleotides deleted fromeach end. In a further embodiment, the ITRs comprise a nucleic acidsequence of SEQ ID NO:1 and/or SEQ ID NO:2. The ITRs may be in the“flip” or “flop” orientation.

The term “permissive cells” means cells in which parvovirus B19 isolatescan be cultured. The permissive cells are eukaryotic cells. Examples ofpermissive cells include, but are not limited to primary erythroidprogenitor cells from bone marrow, blood, or fetal liver cells,megakaryoblast cells, UT7/Epo cells, UT7/Epo-S1 cells, KU812Ep6 cells,JK-1 cells, and MB-O2 cells.

“Percent (%) nucleic acid sequence identity” with respect to the nucleicacid sequences identified herein is defined as the percentage ofnucleotides in a candidate sequence that are identical with thenucleotides in a reference B19 nucleic acid sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. In some embodiments, the reference B19nucleic acid sequence is that of SEQ ID NO:5 or that of SEQ ID NO:24.Alignment for purposes of determining percent nucleic acid sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Thoseskilled in the art can determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full-length of the sequences being compared.

For purposes herein, the % nucleic acid sequence identity of a givennucleic acid sequence A to, with, or against a given nucleic acidsequence B (which can alternatively be phrased as a given nucleic acidsequence A that has or comprises a certain % nucleic acid sequenceidentity to, with, or against a given nucleic acid sequence B) iscalculated as follows:

100 times the fraction W/Z

where W is the number of nucleotides scored as identical matches by thesequence alignment program in that program's alignment of A and B, andwhere Z is the total number of nucleotides in B. It will he appreciatedthat where the length of nucleic acid sequence A is not equal to thelength of nucleic acid sequence B, the % nucleic acid sequence identityof A to B will not equal the % nucleic acid sequence identity of B to A.

“Recombinant” refers to a polynucleotide that has been isolated and/oraltered by the hand of man or a B19 clone encoded by such apolynucleotide. A DNA sequence encoding all or a portion of a B19 viralgenome may be isolated and combined with other control sequences in avector. The other control sequences may be those that are found in thenaturally occurring gene or others. The vector provides for introductioninto host cells and amplification of the polynucleotide. The vectorsdescribed herein for B19 clones are introduced into cells and culturedunder suitable conditions as known to those of skill in the art.Preferably, the host cell is a bacterial cell or a permissive cell.

The term “transformation” as used herein refers to introducing DNA intoa bacterial cell so that the DNA is replicable, either as anextrachromosomal element or by chromosomal integrant. Depending on thehost cell used, transformation is done using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride is generally used for bacterial cells that contain substantialcell-wall barriers. Another method for transformation iselectroporation.

The term “transfection” as used herein refers to introducing DNA into aeukaryotic cell so that the DNA is replicable, either as anextrachromosomal element or by chromosomal integrant. Depending on thehost cell used, transfection is done using standard techniquesappropriate to such cells. Methods for transfecting eukaryotic cellsinclude polyethyleneglycol/DMSO, liposomes, electroporation, andelectrical nuclear transport.

The term “transfection efficiency” as used herein means the percentageof total cells contacted with a nucleic acid, such as a plasmid, thattake up one or more copies of the plasmid. Tranfection efficiency canalso be expressed as the total number of cells that take up one or morecopies of the plasmid per μg of plasmid. If the plasmid contains areporter gene, transfection efficiency of cells can also be expressed inunits of expression of the reporter gene per cell.

The term “replicable vector,” as used herein, is intended to refer to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked into a cell and providing for amplification ofthe nucleic acid. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome. Inthe present specification, “plasmid” and “vector” may be usedinterchangeably as the plasmid is the most commonly used form of vector.In some embodiments, the vector is a vector that can replicate to highcopy number in a cell.

II. Modes for Carrying Out the Invention

Previous attempts to produce infectious clones of parvovirus B19 havebeen unsuccessful due to deletions in the ITR sequences (Shade et al.,1986, J. Virol., 58:921-936) and the instability of the ITRs inbacterial cells. In addition, parvovirus B19 can be cultured inpermissive cells but the amount of virus produced in these cells is verysmall. There have been no methods or clones of the viral genome that canprovide for consistent production of infectious virus. Utilizing themethods of the invention, the genome of parvovirus B19 isolate wascloned and sequenced. A vector was prepared comprising a B19 viralgenome and the vector was used to clone the viral genome. The parvovirusB19 clone can be introduced into other cells types (whether permissiveor not) to produce infectious virus.

The infectious clone and methods described herein can be utilized in avariety of assays and to develop therapeutic products. The infectiousclone is useful for producing infectious virus. An in vitro system forproducing infectious virus particles can be used in screening methods toidentify agents such as antibodies or antisense molecules that caninhibit viral infectivity or reproduction. The infectious virus and/orinfectious virus in a host cell can be utilized to form immunogeniccompositions to prepare therapeutic antibodies or vaccine components.Antibodies and primers can be developed to specifically identifydifferent parvovirus B19 isolates. The ability to produce infectiousvirus in vitro is also useful to develop attenuated strains of the virusthat may be utilized in vaccines.

A. Methods of the Invention

One aspect of the invention involves a method of cloning a viral genomethat has one or more inverted repeats or secondary structure of nucleicacid that is unstable in cells. A method of the invention comprisesintroducing the viral genome into a bacterial cell that is deficient inrecombinase enzymes such as recA1, end A1, recB, recJ or combinationsthereof. The bacterial cells are incubated at a low temperature, forexample about 25° C. to 35° C., preferably about 25° C. to 32° C., andmore preferably about 28° C. to 31° C., and most preferably about 30° C.The cells are incubated for a time sufficient to allow amplification ofthe viral genome. Preferably, the incubation time is about 8 to 24hours, more preferably about 8 to 12 hours. The viral genome isrecovered from the bacterial cells.

In some embodiments, the methods of the invention include a method forcloning an infectious parvovirus B19 clone. In an embodiment, the methodcomprises introducing a replicable vector comprising a parvovirus B19viral genome or portion thereof into prokaryotic cells that aredeficient in major recombination genes, such as for example recA1,endA1, recB and/or recJ or combinations thereof. The cells are incubatedat a low temperature for a time sufficient to allow amplification of thevector. The infectious clone is recovered from the prokaryotic cells.Once the infectious clone is prepared it can be introduced into othercell types, whether permissive or not, and provide infectious virus.

Preparing a Clone of the Viral Genome

The infectious clone is comprised of all or a portion of a viral genomeof parvovirus B19 and a replicable vector that can provide foramplification of the viral genome in a cell, such as a bacterial cell.In some embodiments, the vector has a bacterial origin of replication.In some embodiments, the vector is a plasmid. In some embodiments, thevector can be selected based on the host cell as well as othercharacteristics such as compatibility with host cell, copy number, andrestriction sites. Vectors that can be used in the invention include,without limitation, pBR322, pProExHTb, pUC19, and pBluescript KS.

The method of cloning a parvovirus genome can be applied to anyparvovirus genome. The parvovirus genome includes those obtained fromknown isolates, those isolated from samples from infected tissues, orparvovirus genomes from any source including those that have beenmodified. All or a portion of the viral genome can be cloned. In someembodiments, the parvovirus B19 genome is a full-length genome. In otherembodiments, a portion of the parvovirus genome comprises or consists ofnucleic acid sequence encoding at least one ITR, VP2, NS and the 11 kDaprotein in a single replicable vector. The portion of the viral genomeis that portion that is sufficient to provide for production ofinfectious virus. In other embodiments, the parvovirus genome comprisesor consists of a nucleic acid encoding an 11R at the 5′ end and an ITRat the 3′ end, VP2, NS and the 11 kDa protein in a single replicablevector. In an embodiment, the B19 genome comprises a polynucleotideencoding an infectious B19 clone having at least 90% nucleic acidsequence identity with SEQ ID NO:5 and/or SEQ ID NO:24. In anotherembodiment, the B19 genome comprises a nucleic acid sequence of SEQ IDNO:5.

The parvovirus B19 genome preferably comprises one or more ITRsequences. The ITRs include an imperfect palindrome that allows for theformation of a double stranded hairpin with some areas of mismatch thatform bubbles. The ITRs serve as a primer for viral replication andcontain a recognition site for NS protein that may be required for viralreplication and assembling. In some embodiments, the nucleotide sequencethat forms the hairpins is retained and conserved. In some embodiments,the location and number of the bubbles or areas of mismatch areconserved as well as the NS binding site. The NS binding site providesfor cleavage and replication of the viral genome.

In an embodiment, the parvovirus B19 genome comprises one or more ITRsequences. Preferably, the B19 genome comprises an ITR sequence at the5′ end and the 3′ end. An ITR may be about 350 nucleotides to about 400nucleotides in length. An imperfect palindrome may be formed by about350 to about 370 of the distal nucleotides, more preferably about 360 toabout 365 of the distal nucleotides. Preferably the imperfect palindromeforms a double-stranded hairpin. In an embodiment, the ITRs are about383 nucleotides in length, of which about 365 of the distal nucleotidesare imperfect palindromes that form double-stranded hairpins. In anotherembodiment, the ITRs are about 381 nucleotides in length, of which about361 of the distal nucleotides are imperfect palindromes that formdouble-stranded hairpins. In some embodiments, a B19 genome comprises atleast 75% of the nucleotide sequence that forms the hairpin in the ITRat the 5′ end and 3′ end of the genome. In other embodiments, the ITRsmay have 1 to about 5 nucleotides deleted from each end. In preferredembodiments, the ITR has at least about 94%, more preferably 95%, morepreferably 96%, more preferably 97%, more preferably 98%, morepreferably about 99%, and more preferably 100% of the sequence of thatof viral genome isolated from nature, such as that of SEQ ID NO:5 or SEQID:24. In a further embodiment, the ITRs comprise a nucleic acidsequence of SEQ ID NO:1 and/or SEQ ID NO:2. The ITRs may be in the“flip” or “flop” orientation.

The parvovirus genome may have variation due to variation in naturallyoccurring isolates. For example, isolates of parvovirus B19 frominfected tissues can have about 90% sequence identity or greater to thatof parvovirus B19 Au (GeneBank accession number M13178; SEQ ID NO:24) Insome embodiments, a parvovirus genome has at least 90% sequenceidentity, more preferably more preferably at least 91%, more preferablyat least 92%, more preferably at least 93%, more preferably at least94%, more preferably at least 95%, more preferably at least 96%, morepreferably at least 97%, more preferably at least 98%, more preferablyat least 99% or greater sequence identity to that of a parvovirus B19genome comprising a nucleic acid sequence of parvovirus B19 Au GeneBankaccession number M13178; SEQ ID NO:24).

In some cases, alterations or modifications may be made to the nucleicacid sequence of the viral genome of a viral isolate using standardmethods to form variant viral genomes. The alterations may be made toadd or delete characteristics to the nucleic acid sequence. For example,it may be desirable to add or delete a restriction site or add asequence that can serve to identify the viral genome. In a specificembodiment, a vector, identified as pB19-M20 comprises a full-lengthclone of parvovirus B19 having a sequence of SEQ ID NO:5 but with achange at nucleotide 2285 from a cytosine to a thymine, resulting inconversion of BsrI site to a Dde site. In another embodiment, a vector,identified as pB19-4244d comprises a full-length clone of parvovirus B19having a sequence of SEQ ID NO:5 but with a change to eliminate an XbaIrestriction site.

Alternatively it may be desirable to add a nucleic acid sequence thatencodes a heterologous polypeptide to the infectious clone. Such aheterologous polypeptide may include tag polypeptides such aspoly-histidine (poly-His) or poly-histidine-glycine (poly-His-gly) tags;the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol.Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10,G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and CellularBiology, 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoproteinD (gD) tag and its antibody [Paborsky et al., Protein Engineering,3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide[Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitopepeptide [Martin et al., Science, 255:192-194 (1992)]; an “-tubulinepitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166(1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al.,Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)] The heterologouspolypeptides are combined with viral proteins to form fusion proteins.Epitopes from heterologous proteins may be combined with parvovirus B19proteins to form fusion proteins useful for immunogenic compositions.

Preferably, the variant viral genome has at least 90% sequence identity,more preferably at least 91%, more preferably at least 92%, morepreferably at least 93%, more preferably at least 94%, more preferablyat least 95%, more preferably at least 96%, more preferably at least97%, more preferably at least 98%, more preferably at least 99% orgreater sequence identity to that of a parvovirus B19 genome comprisinga nucleic acid sequence of SEQ. ID. NO:5. In some embodiments, theparvovirus genome, preferably has 99.2% sequence identity, morepreferably 99.3%, more preferably 99.4%, more preferably 99.5%, morepreferably 99.6%, more preferably 99.7%, more preferably 99.8%, and morepreferably 99.9% or greater sequence identity to that of a parvovirusB19 genome comprising a polynucleotide sequence of SEQ. M. NO:5.

In some embodiments, the B19 genome is cloned by cloning at least twoportions of the viral genome into separate vectors and recombining thetwo portions into a single vector. Preferably, the two portions of theviral genome comprise an ITR at the end of the portion. The portions ofthe viral genome can be obtained by digesting the genome with arestriction enzyme that cuts the genome at a location between the ITRs.Preferably the restriction enzyme cuts the genome at a location at leastabout 800 nucleotides from the ITR. The portions may be cut andreligated to reduce the vector size and eliminate undesired restrictionsites. For example, the B19 genome may be digested with BamHI. The twofragments (right end genome fragment and left end genome fragment)generated by BamHI digestion are ligated into separate BamHI-StuIdigested pProEX HTb vectors (Invitrogen-Life Technologies). See, forexample, FIG. 3. To reduce the vector size and eliminate undesiredrestriction sites, clones that contain the right end of the genome(pB19-42d6) may be digested with EcoRV and religated. The full-lengthgenome is generated by digesting the plasmid containing the left endgenome fragment (pB19-44) with BamHI and Ecl136II and cloning thefragment containing the left end genome fragment into the BamHI/EheIsite of the pB19-42d6 plasmid (FIGS. 3 and 4).

In some embodiments, it may be desirable to achieve a high efficiency ofligation. In that case, it is preferred that at least about 0.25 μg ofthe viral genome is combined with about 1 μg of the vector, morepreferably about 0.25 to about 0.5 μg of viral genome per 1 μg amount ofvector. The viral genome can be obtained from serum or infected cells.The isolated virus may be high titer virus and/or concentrated toachieve the amount of viral genome necessary for ligation. In someembodiments, the parvovirus B19 isolated from a sample and used toprepare the clone is present in the sample at about 10⁸ to about 10¹⁴genome copies/ml of original sample, more preferably about 10⁸ to about10¹² genome copies/ml of original sample. Virus can be concentrated fromserum or infected cells using standard methods known in the art, such asfor example, velocity and/or equilibrium density centrifugation usingsucrose solutions in low-salt buffer. Preferably, viral genome isconcentrated at about 10⁸ to about 10¹⁴ genome copies/100 μl ofphysiological solution, more preferably about 10⁸ to about 10¹² genomecopies/100 μl of physiological solution.

Introducing and Amplifying a Parvovirus B19 Clone in Prokaryotic Cells

According to the method of cloning a viral genome, a vector comprisingall or a portion of the viral genome is introduced into a prokaryoticcell. Methods of introducing vectors into cells are known to those ofskill in the art and include transformation methods such as calcium saltprecipitation, liposomes, polyethylene glycol/DMSO, electroporation andelectro nuclear transport. In some embodiments, the vector is introducedinto bacterial cells by electroporation.

The bacterial cells are preferably deficient in recombinase enzymes suchas recA1, endA1, recB, recJ, or combinations thereof. In someembodiments, the transformed bacterial cells are preferably E. colicells. In an embodiment, the E. coli cells are McrA-, McrCB-, McrF-,Mrr-, HsdR-, and endA deficient. In another embodiment, the E. colicells comprise a genotype of e14-(McrA-)Δ(mcrCB-hsdSMR-mrr)171 endA1supE44 thi-1 gyrA96 relA1 lac recB recJ sbcC umuC::Tn5 (Kanr) uvrC [F′proAB lacIqZ.M15 Tn10 (Tetr)]; F⁻ mcrA Δ(mrr-hsdRMS-mcrBC) recA1 endA1lon gyrA96 thi-1 supE44 relA1λ⁻ Δ(lac-proAB); or F⁻ mcrAΔ(mcrBC-hsdRMS-mrr) recA1 endA1 lon gyrA96 thi supE44 relA1λ⁻Δ(lac-proAB). In another embodiment, the E. coli cells are cells, suchas for example SURE2® cells (Stratagene, La Jolla, Calif.); Stbl2 cellsor Stble 4 cells (Invitrogen, Carlsbad, Calif.).

In an embodiment, the incubation temperature for the transformedprokaryotic cells is about 25° C. to about 35° C., preferably, about 25°C. to about 32° C., more preferably about 30° C. to about 32° C., morepreferably about 30° C. The prokaryotic cells preferably are platedimmediately following introduction of the viral genome and incubated fora sufficient time to allow for amplification of the viral genome,preferably, from about 12 to about 24 hours, more preferably from about16 to about 18 hours. The clone can be recovered from the cells usingstandard methods. Infectious clones are those that can produce viralDNA, proteins or particles when introduced into other cell types.

Introducing the Infectious Clone into Other Cell Types

Another aspect of the invention, provides a method of producing aninfectious clone or infectious viral particles of parvovirus B19 in aeukaryotic cell. This method can also be utilized to identify and/orconfirm that the parvovirus B19 clone produced is infectious. After theclone is amplified in a bacterial cell and recovered, the infectiousclone may be introduced into other cell types (whether permissive ornot) to identify whether the clone can produce infectious virus or forthe production of infectious virus. The method provides for productionof infectious virus in vitro. Utilizing an infectious clone allowsintroduction of the viral genome into a cell without the need for entrymediated by viral proteins such as the capsid protein. The methodcomprises introducing a vector comprising an infectious clone ofparvovirus B19 or all or a portion of a viral genome into a eukaryoticcell and incubating the cell for a sufficient time to produce infectiousvirus and optionally, detecting production of infectious virus. Themethod of identifying an infectious clone comprises introducing a vectorcomprising all or a portion of a viral genome into a eukaryotic cell;incubating the cell for a sufficient time to produce infectious virus;and detecting production of infectious virus.

In some embodiments, a high efficiency of introduction of the vectorinto eukaryotic cells is desired. Preferably, the method of introductionemployed achieves a transfection efficiency of at least about 15% to100% efficiency, more preferably about 30 to 50% efficiency. The methodis also selected to minimize cytotoxicity to the cells. Preferably,about 20% or greater of the cells are viable and more preferably about50% of the cells or greater. In some embodiments, the vector may be cutwith one or more restriction enzymes to enhance viral replication.

In an embodiment, eukaryotic cells are transfected with an electriccurrent. Methods of transfecting eukaryotic cells utilizing an electriccurrent are known in the art, such as for example, electroporation(Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manual, Vols.1-3, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. or Davis et al.,1986, Basic Methods in Molecular Biology) and electrical nucleartransport (U.S. 20040014220).

In an embodiment, the eukaryotic cells are transfected by electricalnuclear transport. The cells are exposed to an electrical pulsecomprising a field strength of about 2 kV/cm to about 10 kV/cm, aduration of about 10 μsec to about 200 μsec, and a current of at about 1A to about 2.5 A followed by a current flow of about 1 A to about 2.5 Afor about 1 msec to about 50 msec. A buffer suitable for use inelectrical nuclear transport comprises 0.42 mM Ca(NO₃)₂, 5.36 mM KCl,0.41 mM MgSO₄, 103 mM NaCl, 23.8 mM NaHCO₃, 5.64 mM Na₂HPO₄, 11.1 mMd(+) glucose, 3.25 μM glutathione, 20 mM Hepes, and pH 7.3. Followingtransformation, the permissive cells may be incubated for about 10 minat 37° C. before being plated in prewarmed (37° C.) culture medium withserum and incubated at 37° C.

Commercially available devices and buffer systems for electrical nucleartransport, such as for example the AMAXA CELL LINE NUCLEOFECTOR™ system(Amaxa Biosystems Inc., Nattermannallee, Germany; www-amaxa-com), havebeen customized to transduce specific types of eukaryotic cells such as,for example, UT7/Epo cells UT7/Epo-S1 cells. In an embodiment, UT7/Epocells or UT7/Epo-S1 cells are transfected using NUCLEOFECTOR™ reagent Rand program T-20 on the NUCLEOFECTOR™ device according to themanufacturer's instructions (Amaxa Biosystems Inc., Nattermannallee,Germany).

The eukaryotic cells include, but are not limited to, erythroidprogenitor cells, fetal liver cells, UT7/EPO cells, UT7/EPO-S1 cells, orKU812Ep6 cells. In an embodiment, permissive cells include, but are notlimited to, primary erythroid progenitor cells from bone marrow andblood; megakaryoblast cells, fetal liver cells; UT7/Epo cells,UT7/Epo-S1 cells, KU812Ep6 cells, JK-1 and MB-O2. Other eukaryotic celltypes may also be utilized including 293 cells, CHO cells, Cos cells,Hela cells, BHK cells and SF9 cells.

The cells may be incubated in culture medium following introduction ofthe vector comprising a parvovirus B19 viral genome or plated in culturemedium immediately following transfection. The cells may be incubatedfor about 10 min to about 30 min at about 25° C. to about 37° C., morepreferably about 30° C. to about 37° C., more preferably 37° C. beforeplating the cells. Once plated, the cells are incubated under conditionssufficient to provide for production of infectious virus. In someembodiments, the cells are incubated at 37° C. for about 2 to about 4hours, more preferably at least about 6 hours, more preferably at leastabout 12 hours, more preferably at least about 18 hours, more preferablyat least about 24 hours. In an embodiment, the cells are incubated forabout 72 hours post-transfection. Infectious virus particles can beisolated or recovered from cell lysates.

To determine if B19 virus produced by the methods of the invention isinfectious, supernatants prepared from cell lysates of the cells can beused to infect non-transfected cells. In an embodiment, thenon-transfected cells are UT7/Epo-S1 cells. Production of infectious B19virus by the methods of the invention may be detected by analyzing theinfected cells for spliced transcripts of B19 genes. Preferably thespliced transcripts are spliced capsid transcripts encoding, forexample, VP1 or VP2. In an embodiment, infectious B19 is identified bycontacting cells with supernatant from the transformed cells andanalyzing the contacted cells for B19 spliced transcripts. Detection ofspliced capsid transcripts indicates the parvovirus B19 is infectious.Production of infectious B19 virus may be detected by analyzing theinfected cells for B19 viral proteins. Preferably the B19 viral proteinsare capsid proteins, such as for example VP1 and VP2. In an embodiment,infectious parvovirus B19 virus is identified by contacting cells withsupernatant from the transfected cells and analyzing the contacted cellsfor B19 viral proteins. Detection of B19 capsid proteins indicates theparvovirus B19 is infectious. In another embodiment, in vitroneutralization assays can be performed to test whether neutralizingmonoclonal antibodies against parvovirus B19 capsids are able to blockthe infection caused by the cell lysates of transfected cells. Blockingof infectivity by neutralizing antibodies indicates the virus isinfectious.

B. Infectious Parvovirus B19 Clones

The invention also provides infectious B19 clones and polynucleotidesencoding the infectious clones. The infectious clones may be produced bythe methods of the invention. The infectious clone is comprised of allor a portion of a viral genome of parvovirus B19 and a replicable vectorthat can provide for amplification of the viral genome in a bacterialcell. in some embodiments, the vector has a bacterial origin ofreplication. In some embodiments, the vector is a plasmid. In someembodiments, the vector can be selected based on the host cell as wellas other characteristics such as compatibility with host cell, copynumber, and restriction sites. Vectors that can be used in the inventioninclude, without limitation, pBR322, pProExHTb, pUC19, and pBluescriptKS. Preferably, the vector provides for high copy number of theinfectious clone in bacterial cells, eg about 50-100 copies per cell.Several embodiments of the infectious clones are described in theExamples and the Figures.

The method of cloning a parvovirus genome can be applied to anyparvovirus genome. Thus, an infectious clone can comprise a parvovirusgenome obtained from known isolates, those isolated from samples frominfected tissues, or parvovirus genomes from any source including thosegenomes that have been modified. All or a portion of the viral genomecan be cloned. In some embodiments, the parvovirus B19 genome is a fulllength genome. In other embodiments, a portion of the parvovirus genomecomprises or consists of nucleic acid sequence encoding at least oneITR, VP2, NS and the 11 kDa protein in a single replicable vector. Theportion of the viral genome is that portion that is sufficient toprovide for production of infectious virus. In other embodiments, theparvovirus genome comprises or consists of a nucleic acid encoding anITR at the 5′ end and an ITR at the 3′ end, VP2, NS and the 11 kDaprotein in a single replicable vector. In an embodiment, the B19 genomecomprises a polynucleotide encoding an infectious B19 clone having atleast 90% nucleic acid sequence identity with SEQ ID NO:5. In anotherembodiment, the B19 genome comprises a nucleic acid sequence of SEQ IDNO:5.

The parvovirus B19 genome preferably comprises one or more ITRsequences. “ITR” or “ITR sequence” refers to an inverted terminal repeatof nucleotides in a nucleic acid such as a viral genome. The ITRsinclude an imperfect palindrome that allows for the formation of adouble stranded hairpin with some areas of mismatch that form bubbles.The ITRs serve as a primer for viral replication and contain arecognition site for NS protein that may be required for viralreplication and assembling. In some embodiments, the location and numberof the bubbles or areas of mismatch are conserved as well as the NSbinding site. The NS binding site provides for cleavage and replicationof the viral genome. In an embodiment, the parvovirus B19 genomecomprises one or more ITR sequences. Preferably, the B19 genomecomprises an ITR sequence at the 5′ end and the 3′ end. An ITR may beabout 350 nucleotides to about 400 nucleotides in length. An imperfectpalindrome may be formed by about 350 to about 370 of the distalnucleotides, more preferably about 360 to about 365 of the distalnucleotides. Preferably the imperfect palindrome forms a double-strandedhairpin. In an embodiment, the ITRs are about 383 nucleotides in length,of which about 365 of the distal nucleotides are imperfect palindromesthat form double-stranded hairpins. In another embodiment, the ITRs areabout 381 nucleotides in length, of which about 361 of the distalnucleotides are imperfect palindromes that form double-strandedhairpins. In some embodiments, a B19 genome comprises at least 75% ofthe nucleotide sequence that forms the hairpin in the ITR at the 5′ endand 3′ end of the genome. In other embodiments, the ITRs may have 1 toabout 5 nucleotides deleted from each end. In preferred embodiments, theITR has at least about 94%, more preferably 95%, more preferable 96%,more preferably 97%, more preferably 98%, more preferably about 99%, andmore preferably 100% of the sequence of that of viral genome isolatedfrom nature, such as that of SEQ ID NO:5 or SEQ ID:24. In a furtherembodiment, the ITRs comprise a nucleic acid sequence of SEQ ID NO:1and/or SEQ ID NO:2. The ITRs may be in the “flip” or “flop” orientation.

The parvovirus genome may have variation due to variation in naturallyoccurring isolates. For example, isolates of parvovirus B19 frominfected tissues can have about 90% sequence identity or greater to thatof parvovirus B19-Au (GeneBank Accession No. M13178; SEQ ID NO:24). Insome embodiments, a parvovirus genome has at least 90% sequenceidentity, more preferably more preferably at least 91%, more preferablyat least 92%, more preferably at least 93%, more preferably at least94%, more preferably at least 95%, more preferably at least 96%, morepreferably at least 97%, more preferably at least 98%, more preferablyat least 99% or greater to that of a parvovirus B19 genome comprising anucleic acid sequence of parvovirus B19 Au (GeneBank Accession No.M13178; SEQ ID NO:24).

In some cases, alterations or modifications may be made to the nucleicacid sequence of the viral genome using standard methods. Thealterations may be made to add or delete characteristics to the nucleicacid sequence. For example, it may be desirable to add or delete arestriction site or add a sequence that can serve to identify the viralgenome. In a specific embodiment, a vector, identified as pB19-M20comprises a full length clone of parvovirus B19 having a sequence of SEQID NO:5 but with a change at nucleotide 2285 from a cytosine to athymine, resulting in conversion of BsrI site to a Dde site. In anotherembodiment, a vector, identified as pB19-4244d comprises a full lengthclone of parvovirus B19 having a sequence of SEQ ID NO:5 but with achange to eliminate an XbaI restriction site.

Alternatively it may be desirable to add a nucleic acid sequence thatencodes a heterologous polypeptide to the infectious clone. Such aheterologous polypeptide may include tag polypeptides such aspoly-histidine (poly-His) or poly-histidine-glycine (poly-His-gly) tags;the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol.Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10,G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and CellularBiology, 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoproteinD (gD) tag and its antibody [Paborsky et al., Protein Engineering,3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide[Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitopepeptide [Martin et al., Science, 255:192-194 (1992)]; an “-tubulinepitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166(1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al.,Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)]. Heterologouspolypeptides are combined with viral proteins to form fusion proteins.Epitopes from other proteins may be combined with parvovirus B19proteins to form fusion proteins useful as immunogenic compositions.

Preferably, the viral genome has at least 90% sequence identity, morepreferably at least 91%, more preferably at least 92%, more preferablyat least 93%, more preferably at least 94%, more preferably at least95%, more preferably at least 96%, more preferably at least 97%, morepreferably at least 98%, more preferably at least 99% or greater to thatof a parvovirus B19 genome comprising a nucleic acid sequence of SEQ.ID. NO:5. In some embodiments, the parvovirus genome, preferably has99.2% sequence identity, more preferably 99.3%, more preferably 99.4%,more preferably 99.5%, more preferably 99.6%, more preferably 99.7%,more preferably 99.8%, and more preferably 99.9% or greater sequenceidentity to that of a parvovirus B19 genome comprising a nucleic acidsequence of SEQ. ID. NO:5.

In some embodiments, the B19 genome is cloned by, cloning at least twoportions of the viral genome into separate vectors and recombining thetwo portions into a single vector. Preferably, two portions of the viralgenome comprise an ITR at the end of the portion. The portions of theviral genome can be obtained by digesting the genome with a restrictionenzyme that cuts the genome at a location between the ITRs. Preferablythe restriction enzyme cuts the genome at a location at least about 800nucleotides from the ITR. The portions may be cut and religated toreduce the vector size and eliminate undesired restriction sites. Forexample, the B19 genome may be digested with BamHI. The two fragments(right end genome fragment and left end genome fragment) generated byBamHI digestion are ligated into separate BamHI-StuI digested pProEX HTbvectors (Invitrogen-Life Technologies). See, for example, FIG. 3. Toreduce the vector size and eliminate undesired restriction sites, clonesthat contain the right end of the genome (pB19-42d6) may be digestedwith EcoRV and religated. The full-length genome is generated bydigesting the plasmid containing the left end genome fragment (pB19-44)with BamHI and Ecl136II and cloning the fragment containing the left endgenome fragment into the BamHI/EheI site of the pB19-42d6 plasmid (FIGS.3 and 4).

In some embodiments, it may be desirable to achieve a high efficiency ofligation. In that case, it is preferred that at least about 0.25 μg ofthe viral genome is combined with about 1 μg of the vector, morepreferably about 0.25 to about 0.5 μg or greater of viral genome per 1μg amount of vector. The viral genome can be obtained from serum orinfected cells. The isolated virus may be high titer virus and/orconcentrated to achieve the amount of viral genome necessary forligation. In some embodiments, the parvovirus B19 isolated from a sampleand used to prepare the clone is present in the sample at about 10⁸ toabout 10¹⁴ genome copies/ml of original sample, more preferably about10⁸ to about 10¹² genome copies/ml of original sample. Virus can beconcentrated from serum or infected cells using standard methods knownin the art, such as for example, velocity and/or equilibrium densitycentrifugation using sucrose solutions in low-salt buffer. Preferably,viral genome is concentrated at about 10⁸ to about 10¹⁴ genomecopies/100 μl of physiological solution, more preferably about 10⁸ toabout 10¹² genome copies/100 μl of physiological solution.

The infectious clone is preferably stable and can be passaged throughbacterial cell culture without loss of functional ITRs. The stabilitycan be determined by introducing the infectious clone into bacterialcells and subcloning and religating several times. In preferredembodiments, the clone can be passaged in bacterial cells attemperatures ranging from about 30° C. to about 37° C. at least about 10times without substantial loss of ITR nucleic acid sequence.

C. Recombinant Methods, Vectors, and Host Cells

The infectious B19 clones of the invention are produced by synthetic andrecombinant methods. Accordingly, the invention relates topolynucleotides encoding the infectious B19 clones of the invention(such as for example a B19 genome) and host cells containing theinfectious clone, as well as methods of making such vectors and hostcells by recombinant methods.

The B19 clones of the invention may be synthesized or prepared bytechniques well known in the art. Some nucleotide sequences forparvovirus B19 genomes are known and readily available, for example, onthe Internet at GenBank (accessible at www-ncbi-nlm-nihgov/entrez). Thenucleotide sequences encoding the B19 clones of the invention may besynthesized or amplified using methods known to those of ordinary skillin the art including utilizing DNA polymerases in a cell freeenvironment.

The B19 clones of the invention can be produced from viral isolatedobtained from biological samples. The polynucleotides may be produced bystandard recombinant methods known in the art, such as polymerase chainreaction (Sambrook, et al., 1989, Molecular Cloning, A LaboratoryManual, Vols. 1-3, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.Methods of altering or modifying nucleic acid sequences are also knownto those of skill in the art.

As described herein in the methods of the invention, the B19 genome maybe assembled from polymerase chain reaction cassettes sequentiallycloned into a vector containing a selectable marker for propagation in ahost. Such markers include dihydrofolate reductase or neomycinresistance for eukaryotic cell culture and tetracycline or ampicillinresistance genes for culturing in E. coli and other bacteria.

The polynucleotide may be inserted into a replicable vector for cloning(amplification of the DNA) as described in the methods herein. Variousvectors are publicly available. The vector may, for example, be in theform of a plasmid, cosmid, viral particle, or phage. The appropriatenucleic acid sequence may be inserted into the vector by a variety ofprocedures. In general, DNA is inserted into an appropriate restrictionendonuclease site(s) using techniques known in the art. Vectorcomponents generally include, but are not limited to, one or more of asignal sequence, an origin of replication, one or more marker genes, anenhancer element, a promoter, and a transcription termination sequence.Construction of suitable vectors containing one or more of thesecomponents employs standard ligation techniques that are known to theskilled artisan.

Examples of suitable replicable vectors include, without limitation,pCR-Blunt II TOPO vector (Invitrogen, San Diego, Calif.), pProEX Htbvector (Invitrogen, San Diego, Calif.), and pBR332 (Deiss et al., 1990,Virology, 175:247-254), and pBluescipt SK. The polynucleotide can beoperably linked to an appropriate promoter such as, for example, theparvovirus B19 p6 promoter. Additional suitable promoters are known inthe art such as SV40 or CMV. The replicable vectors may further containsites for transcription initiation, transcription termination, and aribosome binding site for translation.

In an embodiment, the full length B19 genome is cloned by digesting thegenome with a restriction enzyme that cuts the genome into twofragments, cloning the two fragments, and religating the two fragmentsto form the full-length genome. The B19 genome may be digested, forexample, with BamHI. The two fragments (right end genome fragment andleft end genome fragment) generated by BamHI digestion are ligated intoseparate BamHI-StuI digested pProEX HTb vectors (Invitrogen-LifeTechnologies). See, for example, FIG. 3. To reduce the vector size andeliminate undesired restriction sites, clones that contain the right endof the genome (pB19-42d6) may be digested with EcoRV and religated. Thefull-length genome is generated by digesting the plasmid containing theleft end genome fragment (pB19-44) with BamHI and Ecl136II and cloningthe fragment containing the left end genome fragment into the BamHI/EheIsite of the pB19-42d6 plasmid (FIGS. 3 and 4).

Introduction of a recombinant vector comprising a B19 genome into a hostcell, such as for example a bacterial cell or eukaryotic cell, can beaffected by calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,electrical nuclear transport, chemical transduction,electrotransduction, infection, or other methods. Such methods aredescribed in standard laboratory manuals such as Sambrook, et al., 1989,Molecular Cloning, A Laboratory Manual, Vols. 1-3, Cold Spring HarborPress, Cold Spring Harbor, N.Y. or Davis et al., 1986, Basic Methods inMolecular Biology. Commercial transfection reagents, such asLipofectamine (Invitrogen, Carlsbad, Calif.) and FuGENE 6™ (RocheDiagnostics, Indianapolis, Ind.), are also available. Preferablytransfection efficiency of the host cells is about 15% or greater, morepreferably about 20% or greater, more preferably about 30% or greater,more preferably about 40% or greater, more preferably about 50% orgreater, more preferably about 70% or greater

In an embodiment, eukaryotic cells are transfected with an electriccurrent. Methods of transfecting eukaryotic cells utilizing an electriccurrent are known in the art, such as for example, electroporation(Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manual, Vols.1-3, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. or Davis et al.,1986, Basic Methods in Molecular Biology) and electrical nucleartransport (U.S. 20040014220).

In an embodiment, a eukaryotic cell is transfected by electrical nucleartransport. The permissive cells are exposed to an electrical pulsecomprising a field strength of about 2 kV/cm to about 10 kV/cm, aduration of about 10 μsec to about 200 μsec, and a current of at about 1A to about 2.5 A followed by a current flow of about 1 A to about 2.5 Afor about 1 msec to about 50 msec. A buffer suitable for use inelectrical nuclear transport comprises 0.42 mM Ca(NO₃)₂, 5.36 mM KCl,0.41 mM MgSO₄, 103 mM NaCl, 23.8 mM NaHCO₃, 5.64 mM Na₂HPO₄, 11.1 mMd(+) glucose, 3.25 μM glutathione, 20 mM Hepes, and pH 7.3. Followingtransformation, the permissive cells may be incubated for about 10 minat 37° C. before being plated in prewarmed (37° C.) culture medium withserum and incubated at 37° C.

Commercially available devices and buffer systems for electrical nucleartransport, such as for example the AMAXA CELL LINE NUCLEOFECTOR™ system(Amaxa Biosystems Inc., Nattermannallee, Germany; www-amaxa-com), havebeen customized to transduce specific types of eukaryotic cells such as,for example, UT7/Epo cells UT7/Epo-S1 cells. In an embodiment, UT7/Epocells or UT7/Epo-S1 cells are transfected using NUCLEOFECTOR™ reagent Rand program T-20 on the NUCLEOFECTOR™ device according to themanufacturer's instructions (Amaxa Biosystems Inc., Nattermannallee,Germany).

D. Uses

The infectious clone and methods described herein can be utilized in avariety of assays and to develop therapeutic products. As discussedpreviously, methods for consistently obtaining infectious virus in cellculture were not previously known. An in vitro system for producinginfectious virus particles can be used in screening methods to identifyagents such as antibodies or antisense molecules that can inhibit viralinfectivity or reproduction. The infectious virus and/or infectiousvirus in a host cell can be utilized to form immunogenic compositions toprepare therapeutic antibodies or vaccine components. Antibodies andprimers can be developed to specifically identify different parvovirusB19 isolates. The ability to produce infectious virus consistently invitro is also useful to produce attenuated virus that may be used in avaccine.

The infectious B19 clones of the invention are useful in diagnosticassays. The presence or absence of an antibody in a biological samplethat binds to a B19 clone of the invention can be determined usingstandard methods. Alternatively, the presence or absence of B19parvovirus in a biological sample can be determined can be determinedusing PCR primers specific for nucleic acids encoding an infectiousclone of the invention to amplify any parvovirus B19 DNA that may bepresent in the sample. Several primers have been described in theExamples. The primers and antibodies can be developed to specificallyidentify different viral isolates based on difference in nucleic acid orprotein sequences.

The infectious B19 clones of the invention are also useful to produceantibodies to parvovirus B19. The antibodies are useful in diagnosticassays for detecting the presence of parvovirus B19 in a biologicalsample. Methods for developing antibodies are described below. Oneaspect of the invention provides a method for screening for parvovirusB19 infection, comprising contacting a biological sample with ananti-parvovirus B19 antibody and assaying the biological sample foranti-parvovirus B19 antibody binding. The antibodies, preferablyrecognize a particular isolate.

The invention also provides methods for screening for antibodies thatmay inhibit or antagonize B19 infection of permissive cells. Theantagonist effect of anti-parvovirus B19 antibodies may determined byanalyzing cells for B19 capsid proteins or B19 spliced capsidtranscripts as described above. Antagonist antibodies can be preparedand screened as described below.

The infectious parvovirus B19 clone and/or host cells comprising theclone can be used as immunogenic compositions to prepare vaccinecomponents and/or to develop antibodies that can be used in diagnosticor other assays. For example, host cell cultures comprising theparvovirus B19 clone can be heat inactivated and used as an immunogen.Passaging of an infectious clone in vitro can provide an attenuatedstrain of parvovirus B19 useful in vaccine compositions.

E. Production of Antibodies

1. Polyclonal Antibodies

Polyclonal antibodies to infectious B19 clones of the invention of theinvention are preferably raised in animals by multiple subcutaneous (sc)or intraperitoneal (ip) injections of the relevant antigen and anadjuvant. The relevant antigen may be, for example, one or more B19clones of the invention or one or more B19 proteins, such as NS, VP1,VP2, 11-kDa protein, 7.5-kDa protein, and/or protein X, derived from aninfectious clone of the infection. It may be useful to conjugate therelevant antigen to a protein that is immunogenic in the species to beimmunized, e.g., keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor using a bifunctional orderivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═NR,where R and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with 1/2 to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

In an alternative embodiment, the animals are immunized with arecombinant adenovirus vector expressing one or more viral proteinsderived from an infectious clone of the invention, such as for exampleVP1 and/or VP2, followed by booster immunizations with the viralproteins. The polyclonal antibodies generated by the immunizations mayundergo a screen for B19 antagonist activity. Preferably, antibodies toan infectious B19 clone of the invention inhibit the negative effect ofB19 on erythocyte production. In an embodiment, antibodies thatspecifically bind a B19 clone encoded by a polynucleotide comprising anucleic acid sequence of SEQ ID NO:5 inhibits infection of permissivecells.

The polyclonal antibodies are also screened by enzyme-linkedimmunoabsorbent assay (ELISA) to characterize binding. The antigen panelincludes NS, VP1, VP2, 11-kDa protein, 7.5-kDa protein, and protein X.Animals with sera samples that test positive for binding to one or moreexperimental antigens in the panel are candidates for use in monoclonalantibody production. The criteria for selection for monoclonal antibodyproduction is based on a number of factors including, but not limitedto, binding patterns against a panel of B19 viral proteins.

2. Monoclonal Antibodies

Monoclonal antibodies to an infectious B19 clone of the invention may bemade using the hybridoma method first described by Kohler et al.,Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S.Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized as described above toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to a B19 clone of the invention or viralproteins derived from a B19 clone of the invention used forimmunization. Alternatively, lymphocytes may be immunized in vitro.Lymphocytes then are fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)).

The hybridoma cells are than seeded and grown in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, parental myeloma cells. For example,if the parental myeloma cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen and HIVEnv. Preferably, the binding specificity of monoclonal antibodiesproduced by hybridoma cells is determined by immunoprecipitation orenzyme-linked immunoabsorbent assay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

The monoclonal antibodies are characterized for specificity of bindingusing assays as described previously. Antibodies can also be screenedfor antagonist activity as described previously.

3. Human or Humanized Antibodies

Humanized forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from a CDR of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, rabbit or nonhuman primate havingthe desired specificity, affinity, and capacity. Useful non-humanantibodies are monoclonal antibodies that bind specifically toparvovirus B19. Useful non-human antibodies also include antibodies thatinhibit B19 infection of permissive cells. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies maycomprise residues that are not found in the recipient antibody or thedonor antibody. These modifications may be made to improve antibodyaffinity or functional activity. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the hypervariable regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FRs are those of a human immunoglobulinsequence. The humanized antibody optionally will also comprise at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech5:428-433 (1994).

Human antibodies that specifically bind and/or antagonize parvovirus B19can also be made using the transgenic mice available for this purpose orthrough use of phage display techniques.

An in vitro system for producing infectious virus particles can be usedin screening methods to identify agents such as antibodies or antisensemolecules that can inhibit viral infectivity or reproduction. Ascreening method comprises introducing the viral genome of an infectiousclone of parvovirus B19 into a cell and contacting the cells with apotential inhibitory agent, and determining whether the inhibitory agentinhibits infectivity or replication of the viral genome in the cells.Methods for detecting infectivity and replication of the viral genomehave been described herein. Potential inhibitory agents includeantibodies and anti sense molecules.

The ability to produce infectious parvovirus in vitro may allow for thedevelopment of a vaccine or vaccine components. A vaccine can becomprised of heat inactivated virus or attenuated virus. Inactivatedvirus can be prepared from production of infectious clones using methodsknown to those of skill in the art. Attenuated virus can be obtained byserially passaging the virus under conditions that make the virus nonpathological to humans. The attenuated virus is preferably passagedthrough a cell and under certain conditions that provide for an alteredvirus that is less pathological to humans. Vaccine components can alsoinclude one or more of the parvovirus proteins or parvovirus proteinscombined with epitopes from other infectious agents.

All publications, patents, and patent applications cited herein arehereby incorporated in their entirety by reference. The followingexamples are provided for illustrative purposes only, and are in no wayintended to limit the scope of the present invention.

Example 1 Cloning and Sequencing of Parvovirus B19 Isolate J35Introduction

The nucleotide sequence of B19 was originally established by sequencinga viral isolate designated pvbaua obtained from the serum of a childwith homozygous sickle cell disease (Shade et al. 1986, J. Virol., 58:921-936). Subsequently, many B19 isolates have been sequenced bymultiple methods (Erdman et al. 1996, J. Gen. Virol., 77: 2767-2774).Following alignment of the sequences, there is a 6% divergence amongstthe various isolates (Heegaard & Brown, 2002, Clin. Microbiol. Rev., 15:485-505). The single nonstructural protein (NS1) gene is highlyconserved, and the two capsid proteins, VP1 and VP2, occasionally have agreater variability of 2-3% (Hemauer et al. 1996, J. Gen. Virol., 77:1781-1785; Mori et al. 1987, J. Gen. Virol, 68: 2797-2806).

There is no animal model for B19, and virus can only be grown in culturewith difficulty (Heegaard & Brown, 2002). Parvovirus B19 exhibits aselective tropism for erythroid progenitor cells, and can only becultured in primary erythroid progenitor cells from bone marrow, blood,or fetal liver cells, megakaryoblast cells, UT7/Epo cells, UT7/Epo-S1cells, KU812Ep6 cells, JK-1 cells, and MB-O2 cells. (Ozawa et al., 1986;Brown et al., 1991; Yaegashi et al., 1989; Komatsu et al., 1993;Shimomura et al.; 1992 Miyagawa et al., 1999). These series of examplesestablish a method of producing an infectious clone for parvovirus B19.

Methods

Parvovirus B19 (J35) was obtained from the serum of a child with sicklecell anemia undergoing aplastic crisis and sent to NIH for diagnosticpurposes. The serum was found by dot blot assay (Nguyen et al., 2002) tocontain approximately 10¹² genome copies of B19/mL. UT7/Epo-S1 cells(Shimomura et al., 1992) (maintained in Iscove's modified Dulbecco'smedium (IMDM) containing 10% fetal calf serum, 2 U/ml recombinant humanerythropoietin (Amgen, Thousand Oaks, Calif.), and antibiotics at 37° C.in 5% CO₂) were infected with the J35 serum containing high titer B19virus (Nguyen et al., 2002). DNA was extracted by the DNeasy® method(Qiagen Inc, Valencia, Calif.) and eluted into 100 μl of water.

To obtain the coding region of B19 genome, the primer B19-187FR(CGCTTGTCTTAGTGGCACGTCAAC) (SEQ ID NO:16) was designed from the hairpinregion of the virus using sequences available in GenBank (19-HV;AF162273) (SEQ ID NO:17). High fidelity long PCR amplification wasperformed using the single primer B19-187FR with the HF-2 polymerase kit(BD Biosciences, Palo Alto, Calif.) with 25 cycles of amplification (94°C., 15s; 55° C., 30 s; 72° C. 4 min; followed by 72° C. extension for 7min). The amplicon was cloned by blunt ligation into a pCR-Blunt IITOPO® (Invitrogen, San Diego, Calif.) and transformed into One Shot® Top10 competent E. coli cells (Invitrogen, San Diego, Calif.).

Colonies were screened by hybridization with a ³²P-random-primed B19probe obtained from pYT103 as previously described for dot blothybridization (Nguyen et al., 2002), and positive clones were confirmedby sequencing the plasmids using BigDye® terminator cycle sequencing(ABI-Perkin Elmer, Foster City, Calif.). The full-length sequences ofboth strands were obtained by primer walking.

To obtain the complete hairpin sequence, primers (Table 1) were designedfrom the cloned sequence and from B19 sequences available in GenBank.PCR amplification was performed using ExTaq polymerase with 30 cycles ofamplification. The PCR products were ligated into PCR2.1 TOPO® by TACloning® (Invitrogen-Life Technologies), Top10 cells transformed, andthe products sequenced as above.

All DNA sequences, and the amino acid sequence of open reading frames,were analyzed using Lasergene® software (DNAStar, Inc., Madison, Wis.).DNA pairwise homology was determined by Lipman-Pearson method with aKtuple of 2, gap penalty of 4, and deletion penalty of 12. Multiplesequence alignments were determined using the MegAlign program, usingthe Clustal method with a gap penalty of 10 and gap length penalty of10.

TABLE 2 List of primer pairs used for PCR SEQ Nucleotide Product ID No.Primer Sequence (5′-3′) (bp) 18 B19-1F CCACGATGCAGCTACAACTT 19 B19-186RGTGAGCGCGCCGCTTGTCTTAGTG 186 20 B19-181F GTGAGCGCGCCGCTTGATCTTAGT 21B19-1372R AACTTCCACTGTGACTACTG 1195 22 B19-181F GTGAGCGCGCCGCTTGATCTTAGT23 B19-4899F AACACCACAGGCATGGATAC 518

Discussion

The complete B19 coding region, including half of each ITR, wasamplified using PCR. Although several plasmids containing the B19 genomewere obtained, only one clone, obtained using the primer B19-187FR, didnot contain deletions. This plasmid, designated as pB19-N8 (FIG. 1), wassequenced, and contained a 4844-nucleotide sequence including the entirecoding region, and 177 nucleotides of the ITR. The nucleotide sequenceof this B19 isolate (J35) had 99.1% identity to that of B19-Au isolate(GenBank M13178) (SEQ ID No:24). The putative NS, VP1 and VP2 capsidproteins had 99.4%, 99.4% and 99.6% homology respectively, at the aminoacid level compared to the B19-Au isolate.

The 135 isolate of B19 has a genome of 5592 nucleotides, possessing ITRsof 381 nucleotides in length. The distal 361 nucleotides of theserepeats were imperfect palindromes that form double-stranded hairpins.This normally exist in two sequence orientations, “flip” or itsreverse-complement “flop”, believed to result from hairpin transferduring replication (Deiss et al., 1990).

The complete sequence analysis of the viral genome (J35) indicates thatboth the 5′ and 3′ ITRs have two sequence configurations (SEQ ID NO:1and SEQ ID NO:2) analogous to the flip and flop formats previouslyreported by Deiss et al. (1990) (FIGS. 2A and 2B; SEQ ID NO:3 and SEQ IDNO:4). Although several base changes within the ITRs were identifiedcompared to the previous published sequence of B19 (Deiss et al., 1990),the size and the positions of the bubbles formed by unpaired nucleotidesin these palindromic sequences are conserved among different B19isolates, suggesting an important role of these structures in the lifecycle of B19 virus. In comparison to the previously reported B19sequence, the hairpin of B19-J35 isolate was shorter by two nucleotidesat both 5′ and 3′ ends, but this deletion does not appear to affectviral replication and infection. Unlike other parvoviruses, the hairpinsof B19 do not appear to form a Y- or T-shape structure at theturnaround.

Example 2

Construction of B19 Clones

Introduction

There has only been one previous report of the intact ITRs of the humanpathogenic parvovirus B19 (Deiss et al., 1990). In Deiss et al., thegenome was cloned in two halves, and the sequence of the ITRs obtained.However, Deiss et al. were not unable to successfully ligate the twohalves of the genome together nor could they confirm that the ITRs werecorrect by functional studies. Other attempts to produce an infectiousclone were also unsuccessful due to deletions in the ITR sequences(Shade et al., 1986) and the instability of the ITRs in bacterial cells.Our attempts to construct a full-length clone by ligating the ITRsequences to pB19-N8 were repeatedly unsuccessful.

In the present examples, we successfully clone the full-length B19genome using low incubation temperatures and Sure®2 competent E. colicells (Strategene, La Jolla, Calif.) that are deficient in majorrecombination genes. B19 packages equal numbers of both positive andnegative DNA strands (Summers et al., 1983) and has a unique BamHIrestriction enzyme site in the genome (Cotmore & Tattersall, 1984).These properties were used to clone the full-length B19 genome in twohalves (FIG. 3). We also tested whether the full-length B19 genome,especially the ITR sequences, would be stable in the plasmid backboneduring the multiple steps of molecular cloning experiments.

Methods

B19 DNA was purified from 50 μl of viremic serum (J35) using the HighPure® Viral Nucleic Acid Kit (Roche, Indianapolis, Ind.) to obtainapproximately 1.5 μg of double stranded B19 DNA. Double stranded viralDNA (0.5 μg) was digested with BamHI and both resulting fragments wereligated into BamHI-StuI digested pProEX HTb vector (Invitrogen-LifeTechnologies). The ligated products were electroporated intoelectrocompetent Sure®2 E. coli cells (Stratagene) using a BTXelectroporator, then the bacteria were immediately plated and incubatedovernight at 30° C. The resultant colonies were screened for inserts. Toreduce the vector size and eliminate undesired restriction sites, clonesthat contained the right end of the genome were digested with EcoRV andreligated (pB19-42d6). The insert of the plasmid, together with theinsert of the left end containing plasmid (pB19-44) was completelysequenced. To create full-length clones, pB19-44 was digested with BamHIand Ecl136II and the fragments containing the left end of the genomewere cloned into the BamHI/EheI site of the pB19-42d6 plasmid resultingin pB19-4244 (FIG. 4).

To test the stability of the plasmid containing full-length B19 genome,pB19-4244 was digested with BamHI and religated, and then transformedinto Sure®2 cells. After incubation at 30° C. overnight, 18 colonieswere picked up from the plates and the bacteria were propagated at 30°C. The plasmids were purified and mapped by restriction digestion withHindIII, BssHII, and SalI. The fragments were then analyzed byagarose-electrophoresis.

Discussion

Of the 192 clones that were analyzed, 5 contained the untruncated 5′ endof the genome, and 2 clones contained the untruncated 3′ end of thegenome. All the untruncated clones had the same “flip” format for theirITRs (FIGS. 2A and 2B).

After ligation of the plasmids together, two identical clones consistingof the full-length B19 genome were selected and designated as B19-36 andpB19-4244; GenBank AY386330; SEQ ID NO: 25 (FIG. 4). These full-lengthclones were sequenced and the sequences of inserts showed 100% identityto the corresponding region in the pB19-N8. The full viral genome was5592 nucleotides long, with terminal repeat sequences of 381 nucleotidesthat formed an imperfect palindrome. In comparison to the previouslypublished sequences (Deiss et al., 1990), and the one unpublishedsequence in GenBank (B19-HV; AF162273) (SEQ ID NO:17), there were twoless nucleotides at the start and end of the genome, resulting in apalindromic sequence of 361. As showed in FIG. 2B, the nucleotidesequences of the flip and flop are slightly different from that reportedby Deiss et al. (1990) but the numbers and positions of the unpairednucleotides in these palindromic sequences are conserved among the twodifferent B19 isolates.

We tested whether the full-length B19 genome, especially the ITRsequences, were able to be stabilized in the plasmid backbone during themultiple steps of molecular cloning experiments. The plasmid pB19-4244was digested with BamHI and religated, and then transformed into Sure®2cells. After incubation at 30° C. overnight, 18 colonies were picked upfrom the plate for purification and mapping by restriction digestion.All of the plasmids tested (18/18) had the correct restriction sites,and there were no deletions in the hairpin sequences. The plasmids wereserially passed and then sequenced to confirm the absence of deletionsin the hairpin sequences. We found no evidence of deletions under theconditions used in the present study.

Example 3 Introduction of Mutations into a B19 Infectious CloneIntroduction

As an experimental control, a second infectious clone was produced. Thisclone was generated to have the same nucleotide sequence as plasmidpB19-4244, except for a single nucleotide substitution to confirm thatthe infectious clone could generate infectious virus. The production ofan infectious clone and the ability to manipulate the plasmid will allowthe genome to be studied more systematically.

Method

A second infectious clone was produced by site directed mutagenesis. Thecytosine at position 2285 (B19-J35 isolate) was replaced with a thymineto generate a recognition site for restriction enzyme DdeI to produce anaturally existing variant of B19 (B19-Wi isolate, GenBank M24682; SEQID NO:26). The full-length plasmid pB19-4244 was cut with NheI and the5′ overhang filled in using T4 polymerase. The linearized plasmid wasredigested with XbaI, the B19 fragment (from nucleotide 1247 to 3423 inthe genome of B19-J35 isolate) ligated into an XbaI-Ecl136II-digestedpBluescriptII® KS+ phagemid vector (Stratagene), and site-specificmutagenesis (C2285T) was performed using the QuikChange® Site-directedMutagenesis Kit (Stratagene) and primers CMCf(CATTTGTCGGAAGCTCAGTTTCCTCCGAAG; SEQ ID NO:27) and CMCr(CTTCGGAGGAAACTGAGCTTCCGACAAATG; SEQ ID NO:28). To eliminate anundesired XbaI restriction site in the vector sequence of the plasmidpB19-4244, the plasmid was digested with Ecl136 II-XhoI enzymes, theXhoI overhang was blunted with T4 polymerase, and the plasmid wasreligated (plasmid pB19-4244d, FIG. 5). The plasmid with the B19fragment containing C2285T mutation was digested with MscI-XbaI and theB19 fragment was ligated into the MscI-XbaI digested pB19-4244d plasmidresulting the pB19-M20 clone (FIG. 6).

Discussion

Although the cloned sequence was 99% identical to the B19-Au sequence,it was observed that there was a single nucleotide difference betweenthe J35 sequence (and B19-Au) and the published sequence of anotherisolate B19-Wi (GenBank M24682) that would convert a BsrI site in J35 toa DdeI site. This site was within the RT-PCR product amplified with theprimer pair of B19-2255 and B19-2543 and could potentially be used todistinguish transcripts, and hence, viruses with the different sequence.We therefore constructed a second plasmid, pB19-M20 (FIG. 6), thatcontained the identical full-length clone, but in which the cytosine atthe nucleotide of 2285 was replaced by thymine (C2285T).

Example 4 Infection of Cells with B19 and Detection of Replicative Formsof B19 in Infected Cells Introduction

During the replication of parvovirus B19, the viral single-stranded DNAis converted to a double-stranded replicative form which has either an“extended” or a “turnaround” form at the terminal regions. Theseintermediate structures provide evidence for viral DNA replication andcan be distinguished by BamHI restriction enzyme digestion (Cotmore &Tattersall, 1984) (FIG. 7).

To test whether the B19 genome inserted in pB19-4244 could be excisedfrom the flanking vector sequences and produce progeny viral DNA, wecompared Southern blot analysis of the DNA purified from the cellstransfected with either plasmids cut with SalI enzyme (which releasesthe full-length B19 genome from the plasmid, FIG. 4) or intact plasmids.Additionally, RT-PCR was used to detect transcripts for viral capsids inRNA recovered from transfected cells. The presence or absence of B19capsid proteins was detected via immunofluorescent microscopy. By theseexperimental methods, the presence, transcription, and expression of thecapsid gene could be confirmed.

Method

The conditions and reagents for transfecting plasmid DNA into UT7/Epo-S1cells were first optimized using the plasmid pEGFP-F (BD Biosciences,Palo Alto, Calif.) that encodes farnesylated enhanced green fluorescentprotein (EGFP). Cells were examined at daily intervals for expression ofEGFP by UV microscopy and by FACS analysis. Conditions that gave themaximum number of cells expressing EGFP with minimum cytotoxicity werechosen. For subsequent experiments UT7/Epo-S1 cells were transfectedusing the AMAXA® Cell Line Nucleofector™ kit R according tomanufacture's instruction (AMAXA Biosystems Inc., Nattermannallee,Germany). The cells were harvested at various times posttransfection andused for DNA, RNA, and immunofluorescence studies. For infectionstudies, cells were harvested at 72 h posttransfection, washed free ofinoculums using fresh culture medium, and cell lysate prepared by threecycles of freeze/thawing. After centrifugation at 10,000 g for 10 min,the clarified supernatant was treated with RNase (final concentration of1 U/μl, Roche Applied Science, Indianapolis, Ind.) and collected forfurther infections.

Total RNA was extracted from the UT7/Epo-S1 cells (2×10⁵) using RNASTAT60™ (Tel-Test Inc., Friendswood, Tex.). Residual DNA was removed byDNAse I treatment (final concentration, 90 U/ml) for 15 min at roomtemperature. RNA was converted to cDNA with random hexamers andSuperScript™ II (Invitrogen), and RT-PCR for the spliced capsidtranscripts was performed with primers B19-1 (5′GTTTTTTGTGAGCTAACTA3′;SEQ ID NO:6) and B19-9 (5′CCACGATGCAAGCTACAACTT3; SEQ ID NO:7) asdescribed in (Nguyen et al., 2002).

To exclude the possibility that the transcripts detected were derivedfrom laboratory contamination of B19 viral RNA, the cDNA derived fromthe pM20-transfected cells were PCR amplified by using a primer pair ofB19-2255 (GGAACCAGTTCAGGAGAATCA; SEQ ID NO:8) and B19-2543(TGGCAGCTACATCGCACCAA; SEQ ID NO:9), which annealed proximal to theregion containing the site of mutagenesis (C2285T). After purificationusing QIAquick® PCR Purification Kit (Qiagen Inc., Valencia, Calif.),the PCR products were digested with DdeI at 37° C. for 2 h.

Immunofluorescence. Infected or transfected cells were harvested andcytocentrifuged (1500 rpm for 8 mins in a Shandon cytospin 2cytocentrifuge). The cells were fixed in acetone:methanol (1:1) at −20°C. for 5 min, washed twice in phosphate buffered saline (PBS) containing0.1% fetal bovine serum, and incubated with a murine anti-B19 capsidprotein monoclonal antibody (521-5D, gift of Larry Anderson, CDC) in PBSwith 10% fetal calf serum for 1 hr at 37° C. After washing the slidestwice in PBS, the slides were incubated with fluorescein isothiocyanate(FITC)-labeled goat anti-mouse IgG antibody (Jackson ImmunoResearchLaboratories, Inc., West Grove, Pa.) in PBS with 10% fetal calf serumand counterstained with Evans Blue for 30 mins at 37° C., washed in PBS,and examined by UV microscopy.

Southern blot analysis of B19 DNA. DNA was extracted from B19 infectedUT7/Epo-S1 cells (5×10⁵) as previously described (Shimomura et al.,1992). Briefly, 5×10⁵ cells were incubated with 100 mM NaCl, 10mMTris-HCl (pH 7.5), 0.5% sodium dodecylsufate (SDS), 5 mM EDTA, and 200μg/ml proteinase K overnight at 37° C. followed by phenol-chloroformextraction. For some experiments high and low-molecular weight DNA wereseparated by the Hirt method (Hirt, 1967). Purified DNA (400 ng) wasdigested with 20 U of BamH I (single cut in B19) or EcoRI (no cut inB19) at 37° C. for 4 h. The fragments were then separated byagarose-electrophoresis, transferred to a nylon membrane (Nylon+,Amersham), and hybridized with a ³²P-random-primed probe of the completeB19 coding region as previously described (Shimomura et al., 1992).

Discussion

The plasmid pEGFP-F was used to optimize the conditions for transfectingUT7/Epo-S1 cells. Although standard electroporation and liposomes werealso tried, the best results were obtained using the AMAXA® Cell LineNucleofector System™. The highest transfection efficiency (˜70%) withminimum cytotoxicity (˜20%) was achieved with reagent R and T-20 programusing 3 μg pEGFP DNA and 2×10⁶UT7/Epo-S1 cells, following themanufacturer's instructions (AMAXA Biosystems Inc., Cologne, Germany).

UT7/Epo-S1 cells were transfected with plasmids pB19-4244, pB19-M20, andpB19-N8 under the same conditions, and harvested at 72 hpost-transfection. The RT-PCR and immunofluorescence assay wereperformed to detect the viral spliced transcripts and capsid proteins.After RT-PCR, two amplicons of 253 by and 133 bp, representing thealternative spliced transcripts of B19 capsid gene, were detected in thecells transfected with either plasmid (FIG. 8). By immunofluorescenceassay, B19 capsid protein was also detected in the transfected cells,with approximately 15% of the cells having a positive signal whentransfected with pB19-4244 and (FIG. 9B) and 5% with pB19-pN8 (FIG. 9C).There was a significant difference in the number of positive cellsbetween the two different plasmid constructs although the same amount ofplasmid DNA was introduced into the cells under identical conditions.Infection with B19 wild-type virus (J35 isolate) gave approximately 20%positive cells (FIG. 9A).

At 72 h posttransfection, the DNA was extracted from the cells andincubated with the restriction endonuclease EcoRI (no cuts in theparvovirus B19 genome) or BamHI (a single cut in the parvovirus genome).As in B19 infection of UT7/Epo-S1 cells, distinct doublets of 1.5 kb and1.4 kb were detected in all the transfected cell samples digested withBamHI, but not in the plasmid controls (FIGS. 10 and 11). Although aportion of the signal for the 4.1 and 1.5 kb bands in FIG. 10 iscontributed by the transfected DNA, the 1.4 kb band is a definitivemarker for viral genome replication. In addition, a band with amolecular size of 5.6 kb, which corresponds to the size of the viral B19genome, was detected in EcoRI-digested DNA from the cells transfectedwith undigested (SalI) plasmid pB19-M20 (FIG. 11). This indicated thatviral progeny DNA was produced because neither the B19 genome nor vectorcontain an EcoRI restriction enzyme site. Although equal amounts of DNAof either SalI-digested plasmid or whole plasmid were introduced intothe cells, the band density of the replication intermediates in thesample of SalI-digested fragment appeared to be stronger. This suggestedthat the replication process was facilitated when the viral genome wasreleased from the vector backbone.

Example 5 Confirmation of B19 Infectious Virus Introduction

To determine if infectious virus were generated from the UT7/Epo-S1cells transfected with plasmid pB19-4244 or pB19-M20, the supernatantfrom the cell lysates was tested for the detection of splicedtranscripts of viral capsid genes by RT-PCR. We also performed in vitroneutralization assays to confirm that the infectivity of the celllysates was mediated by newly synthesized B19 virons. Finally to confirmthat the viral transcripts in the inoculated cells were being generatedfrom the infectious clone and not from laboratory contamination of wildtype J35 virus, we also used the second infectious clone (pB19-M20) thatcarried a DdeI site that was present in other B19 isolates but not inJ35 virus.

Method

For infection studies, 2×10⁴ of UT7/Epo-S1 cells in 10 μl IMDM weremixed with an equal volume of sample or positive control (J35 serumdiluted to contain 10⁸ B19 genome copies) and incubated at 4° C. for 2 hto allow for maximum virus-cell interaction. The cells were then dilutedto 2×10⁵ cells/ml in the culture medium, and incubated at 37° C., in 5%CO₂. Cells were harvested at 3 days post infection and tested forevidence of infection by detection of viral transcripts and proteinexpression. To determine if infectious virus were generated from theUT7/Epo-S1 cells transfected with plasmid pB19-4244 or pB19-M20, thesupernatant from the cell lysates was tested for the detection ofspliced transcripts of viral capsid genes by RT-PCR. Plasmid pB19-N8,which does not contain intact ITRs and should not produce infectiousvirus, was used as a negative control. B19 infected UT7/Epo-S1 cellswere used as a positive control.

In vitro neutralization assays were performed to test whetherneutralizing monoclonal antibodies against parvovirus B19 capsids wereable to block the infection caused by the cell lysates of transfectedcells. The clarified cell lysates prepared from the transfected cellswere mixed with monoclonal antibody A and E (Yoshimoto et al., 1991) ata dilution of 1:10, and incubated at room temperature for 2 h. Theanti-B19 monoclonal antibody A without neutralizing activities was usedas control. The infection studies were performed as described above.

Discussion

As observed previously, following transfection, spliced transcripts weredetected in all the samples including cells transfected with pB19-N8(FIG. 8). Immediately after inoculation of the clarified supernatantinto the UT7/Epo-S1 cells, no RT-PCR product was detected in any of thesample (FIG. 12A), indicating that there was no carry-over of the RNAfrom the transfected cells. At 72 h post-inoculation spliced transcriptswere detected in the samples derived from the cells transfected withpB19-4244 and pB19-M20, but not with pB19-N8 (FIG. 12B), confirming thatthe full-length viral genome containing complete ITRs is essential forgeneration of infectious viral particles. In addition, no viraltranscripts were detected in cells in which the plasmids were directlyincubated with the cells (no electroporation) (FIG. 12B), suggestingthat the detection of transcripts in the cells inoculated withtransfected-cell lysate was due to the production of infectious B19virus from the plasmid.

The infected cultures were also examined for the production ofparvovirus B19 capsid proteins. At 72 h post-inoculation capsid proteinscould be detected in the nuclei and cytoplasm of cells with thesupernatants derived from either B19 infection or pB19-M20 transfection(FIGS. 13A and 13B), but not in the cells inoculated with either pB19-N8cell lysate (FIG. 13C), or directly with plasmid.

We also performed in vitro neutralization assays to confirm that theinfectivity of the cell lysates was mediated by newly synthesized B19virons. Incubation of the cell lysates with neutralizing monoclonalantibody E (Yoshimoto et al., 1991) reduced the infectivity toundetectable levels in the WA testing. In contrast, incubation with asimilar concentration of monoclonal antibody known to benon-neutralizing (monoclonal antibody A) had no effect on infection.This result further supports our infection experiment, indicating thatinfectious viral particles were produced from the cells transfected withthe plasmids containing full-length B19 genome.

Finally to confirm that the viral transcripts in the inoculated cellswere being generated from the infectious clone and not from laboratorycontamination of wild type J35 virus, we constructed the secondinfectious clone (pB19-M20) that carried a DdeI site that was present inother B19 isolates but not in J35 virus. The sequencing analysis of theplasmids constructed in site-specific mutagenesis showed thatfull-length B19 genome including complete ITR was stable during serialpassages in Sure2 bacteria cells, demonstrating the capacity formanipulating and stably passaging the infectious clone. Aftertransfection, the viral transcripts were tested by restriction enzymedigestion for the presence of the artificially generated DdeI site(FIGS. 14 and 15). No DdeI site was present in transcripts generated bywild-type B19-J35 isolate infection. A DdeI was present only intranscripts from cells infected with lysate from pB19-M20-transfectedcells (FIG. 15).

Example 6 Identification of Viral Proteins Involved in B19 InfectionIntroduction

In common with other parvoviruses, B19 has a small (22 nm),nonenveloped, icosahedral capsid packaging a single-stranded DNA. TheB19 genome has approximately 5,600 nucleotides. The ends of the genomeare long inverted terminal repeats (ITR) of 383 nucleotides in length,of which the distal 365 nucleotides form an imperfect palindrome (Deisset al., 1990). Transcription of the B19 viral genome is controlled by asingle promoter p6 that regulates synthesis of nine viral transcripts toproduce one nonstructural protein (NS), two capsid proteins (VP1 andVP2), and two small proteins (11-kDa and 7.5-kDa) of unknown function(St. Amand et al., 1993, Virology, 195:448-455). Additionally, there isa putative open reading frame encoding a functionally unknown smallprotein X (9-kDA).

In order to experimentally define the role of these genes, we utilizedthe infectious B19 clone described in Example 1 to generate knockoutmutants in which the translational start codon for each of the describedviral genes was substituted with a stop codon.

Methods

To knockout expression of VP1, 7.5-kDa protein, or protein X, thetranslational initiation site (ATG) at 5′ of the gene was replaced witha stop codon (TAG). Plasmid pB19-M20/VP1(−) contained a knockoutmutation for VP1. Plasmid pB19-M20/7.5(−) contained a knockout mutationfor 7.5 kDa protein. Plasmid pB19-M20/X(−) contained a knockout mutationfor protein X.

To prepare these knockout plasmids, the full-length plasmid pB19-4244was cut with NheI and the 5′ overhang filled in using T4 polymerase. Thelinearized plasmid was redigested with XbaI, the B19 fragment (fromnucleotide 1249 to 3425 in the genome of B19-J35 isolate) ligated intoan XbaI-Ecl13611-digested pBluescriptII KS+ cloning vector (Stratagene),and site-specific mutagenesis was performed using the QuickchangeSite-directed Mutagenesis Kit (Stratagene). The primers shown in Table 3were used in the site-specific mutagenesis.

TABLE 3 Knockout PCR primers SEQ ID Gene Primer Nucleotide Sequence NOMutation VP1 Forward 5′GCAAAGCTTTGTAGATTTAG SEQ ID A2624T andAGTAAAGAAAGTGGCAAATGGT NO: 29 T2625A GGG3′ Reverse5′CCCACCATTTGCCACTTTCT SEQ ID TTACTCTAAATCTACAAAGCTT NO: 30 TGC3′7.5-kDa Forward 5′GATTTCCCTGGAATTATAGC SEQ ID A2084T and proteinAGATGCCCTCCACCCAGACC3′ NO: 31 T2083A Reverse 5′GGTCTGGGTGGAGGGCATCT SEQID GCTATAATTCCAGGGAAATC3′NO: 32 Protein X Forward 5′AGTCATCATTTTCAAAGTCTSEQ ID A2874T and AGGACAGTTATCTGACCACC3′ NO: 33 T2875A Reverse5′GGTGGTCAGATAACTGTCCT SEQ ID AGACTTTGAAAATGATGACT3′ NO: 34

To eliminate an undesired XbaI restriction site in the vector sequenceof the plasmid pB19-4244, the plasmid was digested with Ecl13611-XhoIenzymes, the XhoI overhang was blunted with T4 polymerase, and theplasmid was religated (plasmid pB19-4244d).

To knockout expression of 11-kDa protein, the third translationalinitiation site (ATG) at 5′ of the 11-kDa protein gene was replaced witha stop codon (TAG). Plasmid pB19-M20/11(−) contained a knockout mutationfor 11-kDa protein.

The full-length plasmid p1319-4244 described in Example 1 was cut withXabI and BbvClI and the B19 fragment (from nucleotide 1247 to 3423 inthe genome of B19-J35 isolate) was ligated into an XbaI-BbvCI-digestedpBluescriptII KS+cloning vector (Stratagene), and site-specificmutagenesis (A4917T, T4918A) was performed using the QuickchangeSite-directed Mutagenesis Kit (Stratagene) and primers of P11(−)F3(5′CACCACAGACATGGATTAGAAAAGCCTGAAGAATTGTGGAC3′; SEQ ID NO:35), andP11(−)R3 (5′GTCCACAATTCTTCAGGCTTTTCTAATCCATGTCTGTGGTG3′; SEQ ID NO:36).Plasmid with the B19 fragment containing both the A4917T and T4918Amutations was digested with XbaI-BbvCI and the fragment was ligated intoXbaI-BbvCI aI digested pB19-4244d plasmid.

To disrupt the expression of NS protein, the full-length plasmidpB19-4244 was cut with AflII (at nucleotide 756 in B19 genome) and the5′ overhang filled in using T4 polymerase. The linearized plasmid wasreligated with T4 ligase, which generated a stop codon and disrupted theopen reading frame of NS. The plasmid was named pB19-M20/NS(−).

To obtain the ITR deletion mutant, the primer B19-187FR (Table 1) wasdesigned from the hairpin region of the virus using sequences availablein GenBank (19-HV; Genbank accession number AF162273). High fidelitylong PCR amplification was performed using the single primer B19-187FRwith a HF-2 polymerase kit (BD Biosciences, Palo Alto, Calif.) with 25cycles of amplification (94° C. for 15 sec; 55° C. for 30 sec; 72° C.for 4 min; followed by extension at 72° C. for 7 min). The amplicon wascloned by blunt ligation into a pCR-Blunt II TOPO (Invitrogen-LifeTechnologies, San Diego, Calif.) and transformed into Top10 cells(Invitrogen-Life Technologies).

Colonies were screened by hybridization with a ³²P-random-primed B19probe obtained from pYT103 as previously described for dotblothybridization, (Nguyen et al., 2002) and positive clones confirmed bysequencing the plasmids using BigDye terminator cycle sequencing(ABI-Perkin Elmer, Foster City, Calif.). The full-length sequences ofboth strands were obtained by primer walking. One clone (pB19-N8)contained a 4844-nucleotide sequence including the entire coding region,and 177 nucleotides of the ITR at both 5′ and 3′ ends (GenBankAY386330).

UT7/Epo-S1 cells were transfected with the B19 variant plasmids usingthe AMAXA Cell Line Nucleofector™ kit R according to the manufacture'sinstructions (AMAXA Biosystems Inc., Cologne, Germany). The cells wereharvested at various times post-transfection and used for DNA, RNA, andimmunofluorescence studies. For infection studies, cells were harvested72 h post-transfection, washed free of inoculums using fresh culturemedium, and cell lysates prepared by three cycles of freeze/thawing.After centrifugation at 10,000 g for 10 min, the clarified supernatantwas treated with RNase (final concentration of 1 U/μl, Roche) andcollected for further infections.

B19 variant transcripts were detected using RT-PCR. Total RNA wasextracted from the UT7/Epo-S1 cells (2×10⁵) using RNA STAT60 (Tel-TestInc., Friendswood, Tex.). Residual DNA was removed by DNAse I treatment(final concentration, 90 U/ml) for 15 min at room temperature. RNA wasconverted to cDNA with random hexamers and SuperScript II and RT-PCR forthe spliced capsid transcripts was performed with primers B19-1 andB19-9 as described in Example 4.

To exclude the possibility the detected transcripts detected werederived from laboratory contamination of B19 viral RNA, cDNA derivedfrom pM20-transfected cells were PCR amplified by using a primer pair ofB19-2255 and B19-2543 (Table 1), which targeted on the region containingthe site of mutagenesis (C2285T). After purified by using QIAquick PCRPurification Kit (Qiagen Inc., Valencia, Calif.), the PCR products weredigested with DdeI at 37° C. for 2 h.

B19 variants were analyzed for capsid protein expression using theindirect fluorescent antibody assay described in Example 4. Infected ortransfected cells were harvested and cytocentrifuged (1500 rpm for 8mins in a Shandon cytospin 2 cytocentrifge). The cells were fixed inacetone:methanol (1:1) at −20° C. for 5 min and washed twice inphosphate buffered saline (PBS) containing 0.1% fetal bovine serum, andincubated with a mouse monoclonal antibody specific to B19 capsidproteins (521-5D, obtained from Dr. Larry Anderson, CDC) or a rabbitpolyclonal antibody to 11-kDa protein in PBS with 10% fetal calf serumfor 1 hr at 37° C.

For double IFA staining, a lissamine rhodamine-labeled goat anti-mouseIgG and fluorescein isothiocyanate-labeled goat anti-rabbit IgG (JacksonImmunoResearch Laboratories, Inc., West Grove, Pa.) were used assecondary antibodies. The B19 variants were then examined for capsidproteins using confocal microscopy (LSM 510, Leica).

Discussion

No infectious B19 virus was detected in cells transfected with NS, VP1,or 11-kDa protein knockout plasmids. As shown in FIGS. 16A-E,immediately following transfection, spliced transcripts were detected incells transfected with pB19-M20 (FIG. 16A), pB19-M20/VP1(−) (FIG. 16B),pB19-M20/11(−) (FIG. 16C), pB19-M20/7.5(−) (FIG. 16D), pB19-M20/X (−)(FIG. 16E), or pB19-N8 (ITR deletion; FIG. 16F). No spliced transcriptswere detected in cells transfected with pB19-M20/NS(−) immediatelyfollowing transfection (FIG. 16A).

Immediately following infection of UT7/Epo-S1 cells with clarifiedsupernatant from the transfected cells, no RT-PCR product was detectedin any of the cells, indicating that there was no carry-over of the RNAfrom the transfected cells (FIGS. 16A-F). Seventy-two hpost-inoculation, spliced transcripts were detected in cells infectedwith supernatant derived from cells transfected with pB19-M20 (FIG.16A), pB19-M20/7.5(−) (FIG. 16E), or pB19-M20/X (−) (FIG. 16E), but notpB19-M20/NS(−) (FIG. 16A), pB19-M20/VP1(−) (FIG. 16B), pB19-M20/11(−)(FIG. 16C), or pB19-N8 (FIG. 16F). The data in FIG. 16 indicated thatknocking out expression of 11-kDa protein, VP1, NS, or ITR reduced theproduction of infectious viral particles to an undetectable level.

Knocking out 11-kDa protein changed the expression and distributionpattern of B19 viral capsid protein (FIGS. 17A-D). In cells transfectedwith wild-type infectious clone, viral capsid protein first appeared inthe host cell nucleus and was transported into the cytoplasm at a latestage of infection. Capsid protein was either evenly distributed orformed fine clusters in the cytoplasm and nucleus. In cells transfectedwith 11-kDa protein knockout plasmids, production of viral capsidprotein was significantly decreased. Viral capsid protein formed roughclusters in the nucleus and could not be transported to cytoplasm (FIGS.17 C and 17D), suggesting 11-kDa protein may be involved in regulationof viral promoter activity or viral capsid transportation.

Taken together, the data in FIGS. 16A-F and FIGS. 17A-D indicated that11-kDa protein may play an important role in replication of B19 andconfirmed that 11-kDa protein, in addition to ITR sequences and VP2, NS,and VP I proteins, is essential for production of infectious particlesof B19 parvovirus.

1. A method for cloning a parvovirus B 19 viral genome comprising:introducing a vector comprising all or a portion of a parvovirus B 19genome into a prokaryotic cell that is deficient in at least onerecombinase enzyme; incubating the cells at about 25° C. to 35° C.; and(c) recovering the vector from the prokaryotic cells.
 2. The method ofclaim 1, wherein the viral genome comprises an inverted terminal repeat(ITR) at the 5′ end of the genome or at the 3′ end of the genome orboth.
 3. The method of claim 2, wherein the ITR comprises a nucleic acidsequence of SEQ II) NO:I.
 4. The method of claim 2, wherein the ITRcomprises a nucleic acid sequence of SEQ ID NO:2.
 5. The method of claim2, wherein the viral genome further comprises a nucleic acid sequenceencoding at least one or all of VP2, nonstructural protein, or 11-1cDaprotein.
 6. The method of claim 1, wherein the viral genome is a fulllength parvovirus B19 genome.
 7. The method of claim 6, wherein the B19genome comprises a nucleic acid sequence that has at least 90% nucleicacid sequence identity to SEQ ID NO:5 or SEQ ID NO:24. 8-21. (canceled)22. A method for producing an infectious virus of parvovirus B19,comprising: introducing a vector comprising an infectious clone ofparvovirus B19 into a population of cells, wherein the vector is presentin at least about 15% of the cells; and incubating the cells underconditions to allow for viral replication.
 23. The method of claim 22,wherein the cells are eukaryotic cells.
 24. The method of claim 22,wherein introducing the vector into the population of cells is conductedby electric current.
 25. The method of claim 24, wherein the cells areexposed to an electrical pulse comprising a field strength of about 2kV/cm to about 10 kV/cm, a duration of at least about usec, and acurrent of at least about 1 A followed by a current flow of about 1 A toabout 3 A for at least 10 msec. 26-36. (canceled)
 37. An isolatedinfectious parvovirus B19 clone comprising all or a portion of aparvovirus B 19 viral genome and a replicable vector.
 38. The isolatedinfectious parvovirus B19 clone of claim 37, wherein the portion of theviral genome comprises an inverted terminal repeat located at a 5′ and a3′ ends of the genome, wherein the inverted terminal repeat comprises anucleic acid sequence of SEQ ID NO:1 and/or SEQ ID NO:2.
 39. Theinfectious parvovirus B19 clone of claim 38, wherein the portion of theviral genome further comprises one or more of 11-kDa protein, VP1, VP2,and nonstructural protein (NS).
 40. The infectious parvovirus B19 cloneof claim 37, wherein parvovirus viral genome is a full-length genome.41. The infectious parvovirus B19 clone of claim 37, wherein theparvovirus B19 viral genome comprises a polynucleotide having at least90% sequence identity to SEQ ID NO:5 or SEQ ID NO:24.
 42. The infectiousparvovirus B19 clone of claim 41, wherein the parvovirus B19 viralgenome comprises a polynucleotide comprising a nucleic acid sequence ofSEQ ID NO:5
 43. The infectious parvovirus clone of claim 37, wherein theclone is stable upon passage in bacterial cells.
 44. A cell comprisingthe infectious clone of parvovirus B19 of claim
 37. 45. A method ofidentifying an infectious clone comprising: introducing a vectorcomprising all or a portion of a viral genome into a eukaryotic cell;incubating the cell for a sufficient time to produce infectious virus;and detecting production of infectious virus.